JPH0530840B2 - - Google Patents

Description

[Detailed description of the invention]

INDUSTRIAL APPLICATION FIELD OF THE INVENTION The present invention relates to lipoproteins. For more details,
This invention relates to a lipoprotein, which is a component of surfactant, which has remarkable intraalveolar surface activity, and a method for producing the same. Prior Art In recent years, in the treatment of respiratory distress syndrome, which has a high mortality rate, emphasis has been placed on methods of replenishing the deficient pulmonary surfactant with substitutes. A substance consisting of sexual lipids, total cholesterol, carbohydrates, and a trace amount of protein (hereinafter abbreviated as TA-546; JP-A-55-
160721) has been reported. On the other hand, pulmonary surfactant substances scattered in the alveoli of mammals, such as substances isolated and collected from sheep lungs by lung lavage method [Pediatric Research 12
Vol. 841, 1978] has also been reported to contain proteins. Problems to be Solved by the Invention However, it is completely unknown what type or composition of protein contained in TA-546 or lung surfactant, and methods for analyzing and quantifying the protein of both substances are not yet known. If you take this into consideration, it is clear that it is only understood as the sum of lipoproteins and various other proteins. As a result of intensive research on the individual components constituting TA-546 and lung surfactants, the present inventors found that, among mammalian lung-derived proteins, lipoproteins in particular are phosphorylated by choline phosphorus such as dipalmitoylphosphatidylcholine. In addition to the three components of acidic phospholipids such as glyceride, phosphatidylglycerol or phosphatidylserine, and fatty acids such as palmitic acid, this is an essential component to exhibit intraalveolar surface activity; Together with fatty acids, lipoproteins are involved in the adsorption and diffusion of choline phosphoglyceride and acidic phospholipids on the air-liquid surface within the alveoli, and are components that promote the effect of lowering surface tension during alveolar contraction. The present invention was achieved based on the knowledge that the intracellular surface tension reducing effect was not expressed at all. Means for Solving the Problems According to the present invention, there is provided a lipoprotein obtained from mammalian lungs or human amniotic fluid, the chemical composition of which consists of a certain proportion of phospholipids, protein, water, and an unknown composition. Lipoproteins are provided. Further, according to the present invention, a method for producing the lipoprotein is provided. The lipoprotein of the present invention is produced as follows. (a) The lungs removed from mammals such as cows, horses, sheep, or pigs are divided into fist-sized pieces, and unnecessary blood vessels, tracheas,
After removing fat bodies and blood, it is shredded using a meat grinder. Next, the obtained lung pieces are brought into contact with a physiological saline solution for 15 to 120 minutes at 0 to 20° C. with stirring, and then filtered by compression to obtain a crude extract. When human amniotic fluid is used as a raw material, an appropriate amount of human amniotic fluid is collected and used as a crude extract. (b) This crude extract was heated at 0 to 10℃ for 8000 to 20000 r.p.
Centrifuge at a rotational speed of m. to obtain a crude precipitate.
Unwanted pieces of lung tissue remaining in the crude precipitate are removed by resuspending the precipitate in an electrolyte solution such as physiological saline and centrifuging it at 500 to 2000 rpm. (c) Next, suspend this crude precipitate in water, and add sodium chloride to adjust the specific gravity of the liquid to 1.10 to 1.20. This prepared solution is centrifuged at 0 to 10°C for 20 to 180 minutes at a rotational speed of 5,000 to 13,000 rpm to separate it into three layers, and the upper emulsion layer is separated. (d) Suspend this emulsified super-thin part in water, and dialyze the resulting suspension against water at 4-10°C for 6-24 hours using a cellophane membrane to obtain a dialysis solution. This dialyzed fluid is then freeze-dried to obtain a crude dry product. (e) Contact the obtained crude dry product with 20 to 200 times the weight of ethyl acetate or acetone at -10 to 10°C,
After stirring for ~60 minutes, insoluble matter is collected. This insoluble matter is dried, then brought into contact with a chloroform-methanol mixture (volume ratio 2:1) of 80 to 200 times its weight, stirred for 10 to 40 minutes, and then extracted. Collect the filtrate. (f) This extraction filtrate is dried under reduced pressure, and the resulting solid residue is mixed with a chloroform-methanol mixture (volume ratio 2:1) of 2 to 15 times the weight, preferably 5 to 8 times the weight.
to 4:1). This solution is subjected to gel filtration through a column using dextran gel as a carrier such as Cephadex LH-20, Cephadex LH-60, or Cephadex G-25 (manufactured by Pharmacia Fine Chemicals), and the void volume fraction is removed. Collect. It is desirable that the dextran gel column used be equilibrated in advance with the same solvent as the above-mentioned mixture for dissolving the solid residue. The volume of the dextran gel column bed should be 600 ml or more per 1 g of solid residue to be gel-filtered, and the length of the dextran gel layer of the column should be 50 cm or more regardless of the column diameter, preferably 80 cm or more.
It is appropriate to make it at least cm. (g) Dry the above void volume fraction under reduced pressure,
Next, this is suspended in water and then freeze-dried to obtain lipoprotein as a light yellowish brown to yellowish brown powder. [Physical and chemical properties of lipoproteins] (i) Molecular weight The molecular weight measured by a method based on SDS-gel electrophoresis (Separate volume Protein Nucleic Acid Enzyme; Biomembrane Experimental Methods (Part 1) p. 230, 1974) is 30,000 to 38,000. . (ii) Chemical composition The chemical composition ratio of this lipoprotein is as shown in Table 1. In the same table, the composition ratio is the weight percentage of each component relative to the total weight of the lipoprotein. The weight of phospholipids is determined by the method of King et al. [Biochemical Journal, Vol. 26, p. 292].
[1932], and the phosphorus content was multiplied by 25. The protein weight was calculated using the Dulley-Grieve method [Analytical Biochemistry, Vol. 64, p. 136, 1975]
2009] and converted into bovine serum albumin. The water weight was measured by the Karl Fischer method. For unknown components, the weight was defined as the difference between the total weight of lipoproteins and the total weight of phospholipid content, protein content, and water determined as described above.

[Table] (iii) Specific optical rotation [α] ²³ _D : -40° to 85° A 1% aqueous sodium dodecyl sulfate solution was used as the measurement solvent, and the sample concentration was 0.1% (W/V).
The measuring instrument is a DIP-180 automatic polarimeter (JASCO
Co., Ltd.) was used. (iv) Absorption spectrum The infrared absorption spectrum and ultraviolet absorption spectrum are as shown in FIGS. 1 and 2. (v) Solubility, etc. Insoluble in chloroform, benzene, methanol, ethanol, dimethyl sulfoxide and water.
Chloroform-methanol mixture (volume ratio 2:
1 to 4:1) at a concentration of 0.1% (W/V). Insoluble even in 0.1N aqueous sodium hydroxide solution. Since this lipoprotein is insoluble in water and water-containing organic solvents, it is not possible to distinguish between acidic, basic, and neutral lipoproteins. (vi) Color reaction Xanthoprotein reaction is positive. However, it is not possible to clearly determine whether the Bieurett reaction is positive or negative. (vii) Alveolar surface tension reducing effect The surface tension reducing effect of lipoproteins on the physiological saline surface was measured using the Wilhelmy method and the method of King et al. [American Journal of Physiology]
Physiology) Vol. 223, p. 715, 1972], no such effect was observed. By mixing the lipoprotein of the present invention having the above-mentioned physicochemical properties with choline phosphoglyceride, acidic phospholipid, and fatty acids, it can be prepared into a surfactant having remarkable intraalveolar surface activity. The mixing ratio is 0.1 for lipoprotein to the total dry weight of surfactant finally obtained.
~10.0% (W/W), choline phosphoglyceride 50.6~85.0% (W/W), acidic phospholipids 4.5~37.6% (W/W), and fatty acids 4.6~24.6% (W/W). W/W). Examples of choline phosphoglycerides include 1,2-dipalmitoylglycero-(3)-phosphocholine (also known as dipalmitoylphosphatidylcholine), 1,2-dipalmitoylglycero-(3)-phosphocholine (also known as dipalmitoylphosphatidylcholine),
-distearoylglycero-(3)-phosphocholine,
1-palmitoyl-2-stearoylglycero-
(3)-phosphocholine or 1-stearoyl-2
-1,2-diacylglycero-(3)-phosphocholine, 1-hexadecyl-2-palmitoylglycero-(3)-phosphocholine or 1-octadecyl-2-palmitoylglycero-, such as -palmitoylglycero-(3)-phosphocholine, etc. (3)-1-alkyl-2-acylglycero-(3)-phosphocholine such as (3)-phosphocholine or 1,2-dialkylglycero-(1,2-dihexadecylglycero-(3)-phosphocholine etc.) 3)-Phosphocholine is suitable. Optical isomers based on the 2nd carbon of the glycerol residue are known for these compounds, but in this surfactant, D
Any of the so-called D and L bodies, which are a mixture of D and L bodies, can be used. In addition to the single choline phosphoglyceride mentioned above, choline phosphoglycerides include 1,2-diacylglycero(3 A mixture of two or more types of )-phosphocholine or a mixture of the mixture and the above-mentioned single product can also be used. As an acidic phospholipid, 1,2-diacyl-sn
-Glycero-(3)-phosphoric acid (also known as L-α-phosphatidic acid), 1,2-diacyl-sn-glycero-
(3)-Phospho-L-serine (also known as phosphatidylserine) 1,2-diacyl-sn-glycero-(3)-phospho-sn-glycerol (also known as phosphatidylglycerol) or 1,2-diacyl-sn -Glycero-(3)-phospho-(1)-L-myo-inositol (also known as phosphatidylinositol) is suitable. In these compounds, the 1st and 2nd positions may be substituted with the same or different acyl groups. The acyl group here preferably has 14 to 24 carbon atoms. Next, as the fatty acids, free fatty acids, alkali metal salts of fatty acids, fatty acid alkyl esters, fatty acid glycerin esters, fatty acid amides, or mixtures of two or more thereof, and furthermore, fatty alcohols or fatty amines are suitable. As used herein, the term "fatty acids" includes fatty alcohols and aliphatic amines. Suitable free fatty acids are palmitic acid, stearic acid or oleic acid, with palmitic acid being preferred. Sodium palmitate or sodium stearate is suitable as the alkali metal salt of fatty acid, ethyl palmitate is suitable as fatty acid alkyl ester, monopalmitine or monostearin is suitable as fatty acid glycerin ester, and palmitic acid amide is suitable as fatty acid amide. be. Hexadecyl alcohol or octadecyl alcohol is suitable as the fatty alcohol, and hexadecylamine is suitable as the aliphatic amine. The above-mentioned choline phosphoglyceride, acidic phospholipid, and fatty acids may be products separated from animals or plants, semisynthetic products, or chemically synthesized products, and commercially available products thereof can be used. The mixing method is to knead the above-mentioned four components as they are and then dry them to form a surfactant, or to dissolve the four components in an organic solvent and mix them, and dry this solution under reduced pressure to form the resulting residue. Any method may be used in which the surfactant is suspended in a suitable suspension solvent and then freeze-dried (hereinafter referred to as the solution method), but the four components in the obtained surfactant are homogeneously suspended in physiological saline. The solution method is preferred because it is easy. As the organic solvent used to mix the four components in the solution method, a chloroform-methanol mixture (volume ratio 2:1 to 4:1) is suitable. As the suspending solvent, water or a water-ethanol mixture (volume ratio 4:1 to 20:1) is suitable, and a water-ethanol mixture is preferred. Suspension is
It is desirable to carry out the reaction at a temperature of 30 to 60°C, preferably 40 to 50°C, for 5 to 60 minutes, preferably 15 to 30 minutes. Due to the manufacturing process, it is unavoidable that a small amount of water remains in the surf actant obtained by this solution method, but the residual weight ratio is 5.0% (W/W) of the total weight.
It is preferable to dry it until it becomes below. When dried to such a degree, residual ethanol becomes undetectable even when a water-ethanol mixture is used. Actions and Effects of the Invention The action and effects of the lipoprotein of the present invention are shown in the surfactant composed of four components obtained as described above (hereinafter abbreviated as four-component SF) and the surfactant composed of three components not containing lipoproteins. (The following three components
It will also be explained by comparing the surface activity of each representative example with SF (abbreviated as SF). The three-component SF is the same as the four-component SF mentioned above, except that it does not contain lipoproteins.
Manufactured using the same method. A comparative test of surface activity was conducted by experimenting with surface tension lowering effect, air-liquid surface diffusion effect, and alveolar space capacity maintenance effect. The experimental procedure was as follows. (i) Surface tension lowering effect Add 1.0 to 2.0 μg of four-component SF and three-component SF to a physiological saline solution having a surface area of 54.0 cm ² per 1 cm ²
The surface tension was measured continuously according to the Wilhelmy method when the surface area was increased or decreased over 2 to 5 minutes within the range of 54.0 to 21.6 cm ² at 37°C, and the maximum and minimum surface tensions were determined. I asked for it. (ii) Air-liquid surface diffusion effect On the liquid surface of physiological saline, 0.8 per cm ² of surface area.
Drop ~1.5 μg of four-component SF and three-component SF,
Immediately after dropping, the surface tension was measured over time using the vertical plate method, and the equilibrium surface tension and the time to reach it were determined. (iii) Alveolar space capacity maintenance effect Using five rabbit fetuses with a gestation period of 27 days, the alveolar space volume under gradual decrease in airway pressure, especially when the airway pressure was 5 cmH ₂ O (airway pressure was 5 cmH ₂ O). This means that the alveoli of the living body are at the critical point of collapse). This measurement was carried out continuously starting 5 minutes after the administration of the four-component SF or the three-component SF using a water manometer connected to the trachea exposed through an incision in the neck of the fetus. Airway pressure is measured using a 2-channel independently driven syringe pump No.940 connected to the trachea.
(manufactured by Harvard, USA). When administering four-component SF and three-component SF, the concentration is 1.0~
The test was performed by directly injecting 0.05 to 0.5 ml of a physiological saline suspension prepared to a concentration of 6.0% (W/V) into the respiratory tract. In the control group, which was measured in the same manner as above except that physiological saline was used instead of the four-component SF or three-component SF suspension, the alveolar space volume at an airway pressure of 5 cmH ₂ O was approximately It was only 2ml/Kg. The results of the comparative tests are shown in Comparative Examples 1 to 4 below.
The compositional contents of the four-component SF and the three-component SF in each comparative example are lipoprotein, choline phosphoglyceride,
Acidic phospholipids and fatty acids were expressed by their combined weights, and moisture was expressed by the difference between the dry total weight of each surfactant obtained and the combined total weight of the four or three components. In addition, the contents of the four components or three components and water relative to the total dry weight are also listed for reference.

【table】

【table】

【table】

【table】

【table】

[Table] From the above Comparative Examples 1 to 4, by blending the lipoprotein of the present invention, an excellent effect of lowering the surface tension of the surf actant can be realized, a shortening of the diffusion time of the surf actant on the air-liquid surface, and a low equilibrium surface. It is clear that the lipoproteins of the present invention enable tension to be exerted and sufficient alveolar space capacity to be secured. Therefore, the lipoprotein of the present invention can be used to improve the air-liquid surface diffusion of surfactant and the alveolar inner surface during alveolar contraction. It is recognized that it is a component that plays an important role in reducing tension. The present invention will be further explained with examples. Example 1 (a) 128.3 kg of removed lung from a cow was washed with water to remove adhering blood, etc., and then it was cut into fist-sized chunks of meat and unnecessary blood vessels, trachea, etc. were removed using scissors. It was removed. This meat mass was shredded using a meat grinder to obtain 120.1 kg of lung pieces. The lung pieces and physiological saline 490 were mixed and stirred at 4°C for 100 minutes, and the resulting mixture was poured into a filter bag and filtered by compression to obtain a crude extract 470. (b) This crude extract was centrifuged at 10,000 rpm to collect a crude precipitate. 100% of the obtained crude precipitate
The tissue was resuspended in physiological saline and centrifuged at 2000 rpm for 10 minutes to remove remaining tissue pieces as a precipitate. The upper suspension was centrifuged again at 10,000 rpm, and the crude precipitate was collected again. (c) The crude precipitate thus obtained was suspended in 85 g of water, and 25.7 kg of sodium chloride was added to adjust the specific gravity of the liquid to about 1.20. Add this adjustment solution to 10,000 r.
Separate into three layers by centrifugation at 0 °C for 50 min at pm.
The upper thin part of the emulsion was collected. (d) This emulsified upper thin part was suspended in distilled water and dialyzed against distilled water using a cellophane membrane to obtain a dialysis solution. When this dialysis fluid was freeze-dried, it weighed 960 g.
A crude dry product was obtained. (e) Ethyl acetate cooled to 5°C to this crude dry product
was added, stirred for 45 minutes, and then filtered under reduced pressure to remove insoluble materials. After drying this insoluble material,
A chloroform-methanol mixture (volume ratio 2:1) 28 was added to this, and after stirring for 30 minutes, the mixture was filtered with a filter paper to obtain an extracted filtrate. The same mixture 28 was added to the filtration residue again, stirred for 30 minutes, and then filtered with filter paper.
A subsequent extraction filtrate was obtained. This operation was repeated once more to obtain a third extraction filtrate. The total amount of extracted filtrates was 82. (f) This extracted filtrate was dried under reduced pressure and weighed 160.4 g.
A solid residue was obtained. This solid residue was divided into six 25.0 g portions and the other operations were performed. First, 25.0 g of each solid residue was dissolved in 170 ml of a chloroform-methanol mixture (volume ratio 2:1).
Next, each of the obtained solutions was equilibrated with the same mixture separately into a Sephadex LH-20 column (diameter 15.5 cm x 90 cm; column bed volume 17.0).
The mixture was eluted with the same mixture at a flow rate of 5 ml/min and subjected to gel filtration, and void volume fractions were collected. The total volume of the void volume fraction obtained through the six operations was 6480 ml. A sterility test using a trace amount of this fraction confirmed that it was sterile, so the following operations were carried out under sterile conditions. (g) The void volume fraction was dried under reduced pressure, and then suspended in sterile water and freeze-dried to obtain 8.3 g of lipoprotein as a pale yellowish brown powder. The molecular weight is 34000, and the specific rotation [α] ²³ _D is −
It was 69°. In addition, the chemical composition is phospholipid content 62.1
% (W/W), protein content 31.3% (W/W), moisture
4.6% (W/W) and unknown composition 2.0% (W/W)
W) It was. Example 2 (a) 15 kg of removed cow lung was subjected to the same operation as described in step (a) of Example 1 to obtain 13.7 kg of lung fragments. This was added to physiological saline solution 68 and heated to 8℃ for 60 minutes.
After stirring for a minute, the resulting liquid was poured into a filter bag and filtered by compression to obtain a crude extract 61. (b) This crude extract was centrifuged at 12,000 rpm to collect a crude precipitate. Physiological saline 11 was added to the obtained crude precipitate and suspended, and then heated at 4°C for 1500 r.
It was centrifuged at a rotational speed of pm to remove remaining unnecessary tissue pieces as a precipitate. The upper suspension is
It was centrifuged at 12000 rpm and the crude precipitate was collected again. (c) This crude precipitate was suspended in 10 ml of water, 2.23 kg of sodium chloride was added thereto to adjust the specific gravity of the liquid to approximately 1.15, and the adjusted liquid was then centrifuged at 8000 rpm for 60 minutes at 10°C. The upper thin part of the emulsion was collected. (d) This emulsified upper thin part was suspended in distilled water and dialyzed against the distilled water using a cellophane membrane.The resulting dialyzed solution was then freeze-dried to obtain 79 g of a crude dry product. (e) Add acetone 8 to this crude dry product at 4°C,
After stirring for 60 minutes, filter paper filtration was performed to remove insoluble matter. After drying, this insoluble material was brought into contact with a chloroform-methanol mixture (volume ratio 2:1)14.
Stir for 40 minutes, then filter with filter paper to extract the extracted filtrate.
Got 13.8. (f) This extraction filtrate was dried under reduced pressure to obtain 20.5 g of solid residue.
I got it. The obtained solid residue was dissolved in 120 ml of a chloroform-methanol mixture (volume ratio 5:2) and applied to a Sephadex LH-20 column (diameter 15.5 cm x 88 cm; column bed volume 16.6) equilibrated with the same mixture. did. This column was eluted with the same mixture at a flow rate of 1 ml/min and subjected to gel filtration, and 915 ml of void volume fraction was collected.
When a sterility test was conducted using a trace amount of this fraction, it was confirmed that it was sterile, so the following steps were carried out under sterile conditions. (g) Dry the above void volume fraction under reduced pressure,
This was suspended in sterile water and freeze-dried to obtain 1.5 g of yellow-brown lipoprotein powder. The molecular weight is
34000, specific optical rotation [α] ²³ _D is -40°, and the chemical composition is 70.2% (W/W) phospholipid and protein.
23.4% (W/W), moisture 4.0% (W/W), and unknown composition 2.4% (W/W). Example 3 (a) 36 kg of isolated pig lung was subjected to the same operation as described in step (a) of Example 1 to obtain 33.5 kg of lung fragments. Add this to physiological saline solution 168 and heat at 10℃.
After stirring for 30 minutes, the resulting liquid was poured into a filter bag and filtered by compression to obtain crude extract 145. (b) This crude extract was centrifuged at 14000 rpm to obtain a crude precipitate. Physiological saline solution is added to the obtained crude precipitate.
20 and suspended at 1000r.pm at 4℃.
The sample was centrifuged at a rotational speed of 100 mL to remove remaining unnecessary tissue fragments as a precipitate. The upper suspension is
It was centrifuged at 13000 rpm and the crude precipitate was collected again. (c) This crude precipitate was suspended in water 26, and 5.8 kg of sodium chloride was added thereto to adjust the specific gravity of the liquid to approximately 1.15.Then, this adjusted liquid was centrifuged at 8000 rpm for 30 minutes at 4°C. The upper thin part of the emulsion was collected. (d) The emulsified upper thin part was suspended in distilled water and dialyzed against the distilled water using a cellophane membrane, and the resulting dialyzed solution was then freeze-dried to obtain 163 g of a crude dry product. (e) Add acetone 15 to this crude dry product at 6℃,
After stirring for 30 minutes, filter paper filtration was performed to remove insoluble matter. After drying, this insoluble material was brought into contact with a chloroform-methanol mixture (volume ratio 2:1)23.
Stir for 20 minutes, then filter with filter paper to extract the extracted filtrate.
Got 22. (f) This extracted filtrate was dried under reduced pressure to obtain 46.2 g of solid residue.
I got it. 20.0 g of the obtained solid residue was added to 120 ml of a chloroform-methanol mixture (volume ratio 3:1).
Sephadex LH-20 column (diameter 15.5cm x 88cm;
The column bed volume was 16.6). This column was eluted with the same mixture at a flow rate of 3 ml/min and subjected to gel filtration, and a void volume fraction of 910 ml was collected. A sterility test using a trace amount of this fraction confirmed that it was sterile, so the following operations were performed under sterile conditions. (g) Dry the above void volume fraction under reduced pressure,
This was suspended in sterile water and freeze-dried to obtain 1.1 g of yellow-brown lipoprotein powder. The molecular weight is
32000, the specific optical rotation [α] ²³ _D is -51°, and the chemical composition is 69.2% (W/W) phospholipid and protein.
26.4% (W/W), moisture 3.0% (W/W), and unknown composition 1.4% (W/W). Example 4 (a) 15 kg of excised horse lung was subjected to the same operation as described in step (a) of Example 1 to obtain 13.1 kg of lung fragments. This was added to physiological saline solution 65°C and heated to 4°C for 30°C.
After stirring for a minute, the resulting liquid was poured into a filter bag and filtered by compression to obtain a crude extract 56. (b) This crude extract was centrifuged at 13000 rpm to obtain a crude precipitate. Physiological saline solution 9 was added to the obtained crude precipitate and suspended, and then heated at 1500 rpm at 4°C.
The sample was centrifuged at a rotational speed of 100 mL to remove remaining unnecessary tissue fragments as a precipitate. The upper suspension is
It was centrifuged at 10,000 rpm and the crude precipitate was collected again. (c) This crude precipitate was suspended in 10 g of water, and 2.86 kg of sodium chloride was added to adjust the specific gravity of the liquid to approximately 1.20.Then, this adjusted liquid was centrifuged at 8000 rpm for 60 minutes at 4°C. The upper thin part of the emulsion was dispersed. (d) This emulsified upper thin part was suspended in distilled water and dialyzed against the distilled water using a cellophane membrane.The resulting dialyzed solution was then freeze-dried to obtain 76 g of a crude dry product. (e) Ethyl acetate 8 was added to this crude dry product at 10°C, stirred for 60 minutes, and then filtered with a filter paper to remove insoluble matter. After drying, remove the insoluble matter with chloroform.
The mixture was brought into contact with a methanol mixture (volume ratio 2:1) 14 and stirred for 20 minutes, and then filtered with a filter paper to obtain an extracted filtrate 13.1. (f) This extraction filtrate was dried under reduced pressure to obtain 18.1 g of solid residue.
I got it. The obtained solid residue was dissolved in 120 ml of a chloroform-methanol mixture (volume ratio 2:1) and applied to a Sephadex LH-20 column (diameter 15.5 cm x 88 cm; column bed volume 16.6) equilibrated with the same mixture. did. This column was eluted with the same mixture at a flow rate of 1 ml/min and subjected to gel filtration, and 880 ml of void volume fraction was collected.
When a sterility test was conducted using a trace amount of this fraction, it was confirmed that it was sterile, so the following steps were carried out under sterile conditions. (g) Dry the above void volume fraction under reduced pressure,
This was suspended in sterile water and freeze-dried to obtain 0.8 g of yellow-brown lipoprotein powder. The molecular weight is
38000, specific optical rotation [α] ²³ _D is -85°, and the chemical composition is phospholipid content 48.5% (W/W), protein content
48.0% (W/W), moisture 1.8% (W/W), and unknown composition 1.7% (W/W). Example 5 (a) 15 kg of removed sheep lung was subjected to the same operation as described in step (a) of Example 1 to obtain 13.4 kg of lung fragments. Add this to 70% physiological saline solution and store at 4°C for 60%
After stirring for a minute, the resulting liquid was poured into a filter bag and filtered by compression to obtain a crude extract 65. (b) This crude extract was centrifuged at 14000 rpm to obtain a crude precipitate. Physiological saline solution 9 was added to the obtained crude precipitate to suspend it, and then the mixture was heated at 1000 rpm at 4°C.
The sample was centrifuged at a rotational speed of 100 mL to remove remaining unnecessary tissue fragments as a precipitate. The upper suspension is
It was centrifuged at 10,000 rpm and the crude precipitate was collected again. (c) This crude precipitate was suspended in 10 g of water, and 2.86 kg of sodium chloride was added to adjust the specific gravity of the liquid to approximately 1.20.Then, this adjusted liquid was centrifuged at 8000 rpm for 60 minutes at 4°C. The upper thin part of the emulsion was collected. (d) This emulsified upper thin part was suspended in distilled water and dialyzed against the distilled water using a cellophane membrane.The resulting dialyzed solution was then freeze-dried to obtain 71 g of a crude dry product. (e) Ethyl acetate 8 was added to this crude dry product at 10°C, stirred for 60 minutes, and then filtered with a filter paper to remove insoluble matter. After drying, remove the insoluble matter with chloroform.
It was brought into contact with a methanol mixture (volume ratio 2:1) 14 and stirred for 30 minutes, and then filtered with a filter paper to obtain an extracted filtrate 13.7. (f) This extracted filtrate was dried under reduced pressure to obtain 17.5 g of solid residue.
I got it. The obtained solid residue was dissolved in 120 ml of a chloroform-methanol mixture (volume ratio 2:1) and applied to a Sephadex LH-20 column (diameter 15.5 cm x 88 cm; column bed volume 16.6) equilibrated with the same mixture. did. This column was eluted with the same mixture at a flow rate of 1 ml/min and subjected to gel filtration, and 860 ml of void volume fraction was collected.
A sterility test using a trace amount of this fraction confirmed that it was sterile, so the following operations were performed under sterile conditions. (g) Dry the above void volume fraction under reduced pressure,
This was suspended in sterile water and freeze-dried to obtain 0.7 g of yellow-brown lipoprotein powder. The molecular weight is
30000, specific optical rotation [α] ²³ _D is -79°, and the chemical composition is phospholipid content 47.9% (W/W), protein content
44.8% (W/W), moisture 5.0% (W/W), and unknown composition 2.3% (W/W). Example 6 (a) 9.6 ml of human amniotic fluid from 25 people was collected and used as a crude extract. (b) This crude extract was centrifuged at 14000 rpm to obtain a crude precipitate. Physiological saline solution is added to the obtained crude precipitate.
After adding 230 ml and suspending, incubate at 1500 r.p. at 4°C.
Centrifugation was performed at a rotational speed of m. to obtain an upper layer suspension.
This upper layer suspension was centrifuged at 12000rpm,
The crude precipitate was re-collected. (c) This crude precipitate was suspended in 50 ml of water, 14.3 g of sodium chloride was added thereto to adjust the specific gravity of the liquid to approximately 1.20, and the adjusted liquid was then centrifuged at 8000 rpm for 60 minutes at 4°C. The upper thin part of the emulsion was collected. (d) This emulsified upper thin part was suspended in distilled water and dialyzed against the distilled water using a cellophane membrane.The resulting dialyzed solution was then freeze-dried to obtain 2.3 g of a crude dry product. (e) 230 ml of ethyl acetate was added to this crude dry product at 8°C, stirred for 30 minutes, and then filtered with a filter paper to remove insoluble matter. After drying, remove the insoluble matter with chloroform.
The mixture was brought into contact with 400 ml of a methanol mixture (volume ratio 2:1) and stirred for 30 minutes, and then filtered through a filter paper to obtain 390 ml of an extracted filtrate. (f) The extracted filtrate was dried under reduced pressure to obtain a solid residue of 680 mg.
I got it. The obtained solid residue was dissolved in 4 ml of a chloroform-methanol mixture (volume ratio 3:1) and applied to a Sephadex LH-20 column (diameter 2.48 cm x 90 cm; column bed volume 435 ml) equilibrated with the same mixture. did. This column was eluted with the same mixture at a flow rate of 0.2 ml/min and subjected to gel filtration, and 24 ml of void volume fraction was collected. A sterility test using a trace amount of this fraction confirmed that it was sterile, so the following operations were performed under sterile conditions. (g) Dry the above void volume fraction under reduced pressure,
This was suspended in sterile water and freeze-dried to obtain 25 mg of yellow-brown lipoprotein powder. Molecular weight is 36000,
The specific optical rotation [α] ²³ _D is −63°, and the chemical composition is 66.3% phospholipid (W/W) and 28.9% protein.
(W/W), moisture content was 2.8% (W/W), and unknown composition was 2.0% (W/W). Note that the moisture content in this example was measured by a trace moisture measurement method.

[Brief explanation of the drawing]

Figure 1 shows the infrared absorption spectrum of the lipoprotein of the present invention by the KBr tablet method, and Figure 2 shows the infrared absorption spectrum of the lipoprotein of the present invention.
1.37 mg in 10 ml of 1% sodium dodecyl sulfate aqueous solution
The figure shows an ultraviolet absorption spectrum measured using a solution obtained by dissolving .

Claims (1)

[Scope of Claims] 1. A lipoprotein obtained from mammalian lungs or human amniotic fluid, with a composition ratio of phospholipid content of 47.9 to 70.2% (W/W) and protein content of 23.4% to the total weight.
~48.0% (W/W), moisture 1.8~5.0% (W/W)
and unknown composition is 1.4 to 2.4% (W/W),
A lipoprotein characterized by a molecular weight of 30,000 to 38,000. 2 (a) a step of contacting mammalian lung fragments with a physiological saline solution or collecting human amniotic fluid to obtain a crude extract; (b) a step of centrifuging the crude extract to obtain a crude precipitate; (c) adding sodium chloride to the aqueous suspension of the crude precipitate to adjust the specific gravity, centrifuging the resulting adjusted solution to separate the emulsified upper thin part; (d) the milk A step of dialyzing the aqueous suspension of the upper thin part to obtain a dialysis solution, and then freeze-drying this to obtain a crude dry product, (e) bringing the crude dry product into contact with ethyl acetate or acetone to make it insoluble. (f) collecting a void volume fraction from the extract by gel filtration using a dextran gel; (g) A method for producing lipoproteins, which comprises the steps of drying the void volume fraction under reduced pressure and freeze-drying an aqueous suspension of the obtained solid residue.