KR100190826B1 - A pharmaceutical composition for inhibiting haemolysis and its preparation method - Google Patents

Abstract

The present invention relates to an anti-hemolytic agent comprising a sulfolipid with a negative charge of the general formula (I).

LR-SO ₃ X (1)

In the above formula

L is the lipid moiety (the lipid moiety has a sterol structure such as two hydrocarbon chains or cholesterol);

R is a carbohydrate moiety (selected from the group consisting of glocos, galactose, mannose, ribose, 2-deoxyribose, and combinations of two or more thereof);

X is hydrogen or cation (Li, Na, K, 1/2 Mg, 1 / 2Ca) contained in normal blood.

Description

Pharmaceutical composition for inhibiting erythrocyte hemolysis, and preparation method thereof

The present invention relates to a glycolipid composition having a negative charge associated with preventing, delaying and inhibiting red blood cell hemolysis, and a method for preparing the same. Moreover, the present invention relates to the use of negatively charged bile lipids for the treatment of erythrocyte hemolysis, and particularly applies to the treatment of hemolysis by botte induced hemolysis and other etiologies in mammals. It is also applied as an anti-hemolytic composition together with drugs having a hemolytic effect.

Many diseases and disorders associated with red blood cell hemolysis have been known. For example, hemolytic disease of the newborn, paroxysmal nocturnal hematuria, and disease caused by a blood transfusion mistake. Toxins of some types have been known to cause hemolysis, for example, staphylococcus toxin, snake toxins, insect toxins, and the like. In addition, drug-induced immune hemolysis has been known, and hemolysis may be caused by storage of blood or storage of blood components. Because of these hemolytic properties, many drugs are limited in their use.

Erythrocytes are a special system that inhibits hemolysis. The erythrosite membrane contains a special glycoprotein MACIF, and this glycoprotein has been known to have a function of inhibiting hemolysis. In European Patent A2 0 394 035 such a soluble formulation of MACIF is known. Another hemolytic inhibitor glycoprotein is known as _S CR1, and the complement of this receptor type is present in the membrane of blood cells. This glycoprotein is a combination of complement compounds C3b and C4b, wherein the molecular weight is 250 ~ 290 kDa. A method for preparing glycoprotein is already disclosed in PCT / US 93/07406.

The present invention has invented a new biological use in which negatively charged sulfolipid (SL) prevents, inhibits and inhibits hemolysis caused by complement-induced hemolysis and other causes. SL is a term for including both natural products and SL derivatives by artificial synthesis, and is represented by the following general formula (I).

LR-SO ₃ X (I)

In formula (I), L is the lipid moiety, R is the carbohydrate moiety, and X is the hydrogen or cation (Li, Na, K, 1/2 Mg, 1/2 Ca) contained in normal blood.

The lipid moiety has a sterol structure such as two hydrocarbon chains or cholesterol. The lipid moiety is 1,2-diacylglycerol or 1,2-dialkylglycerol, or 1-alkyl-2-acylglycerol, or 1-acyl-2-alkylglycerol having C ₁₂ -C ₁₆ acyl and alkyl chains. Or ceramide. The carbohydrate moiety is selected from the group consisting of glucose, galactose, mannose, ribose, 2-deoxyribose, and combinations of two or more thereof.

Such sulfolipids are natural products or derivatives of natural products, or artificial chemical compounds, for example, cerebroside-3-sulfate, sulfolactosyl ceramides, sulfogalactosyl diacylglycerols, sulfogalactosylalkylacylglycerols, Preferred examples of sulfolipides are cerebroside sulfates and are natural products, derivatives of natural products, chemical compounds.

We found that sulfolipids have a strong antihemolytic effect in complement-induced hemolysis, liposomes or fat emulsions containing sulfolipids with an effective concentration of 50 (IC ₅₀ ) of 3 to 170 μg / ml are preferred. Preference is given to containing 16-90% sulfolipids in the total polar lipids. If the amount of sulfolipids in the total polar lipids is less than 16%, they are not sufficiently antihemolytic. Preferred lipids are mixtures of sulfolipids, phosphatidylcholine, cerebrosides and cholesterol.

Method for producing an anti-hemolytic composition according to the present invention is as follows.

The whole process is divided into two stages: the first stage is to obtain a lipid mixture containing sulfolipid (SL) and the second stage is to make a lipid suspension from isotonic solution. Although there are various methods for obtaining the composition of the present invention, a method of separating lipids from natural products having an optimal amount of SL and an optimal lipid environment is a priority. For example, if you have a commercially available sulfolipid, phosphatidylcholine, cerebroside, and cholesterol, and the composition ratio is 16 to 90% of sulfolipid, 10 to 74% of phosphatidylcholine, 10 to 40% of cerebroside, and 0 to 50% of cholesterol High activity.

However, this method is not suitable for the high price of pure lipids. It is more preferable to obtain a lipid composition containing sulfolipid from the animal brain or spinal cord due to the difference in solubility in organic solvents. The method is described as follows.

1. Finely crush fresh spinal cord with a tissue grinder and add acetone. After stirring, the mixture was filtered to remove the acetone solution.

2. Add acetone to the residue, stir and remove the acetone dissolved material and acetone solution.

3. Add methanol to the residue, stir at room temperature, filter and collect the extracted methanol solution.

4. Add hexane to the residue, stir at room temperature, filter and extract the extracted hexane solution.

5. Distillation under reduced pressure of the extract collected in No. 3 and No. 4.

6. Methylene chloride is added to the residue to dissolve it, and then acetone is added. The mixture is left to settle.

7. Methylene chloride is added to the precipitate to dissolve it, and then methanol is added. The mixture is distilled under reduced pressure to 1/3 volume, and left to settle.

8. Methylene chloride is added to the precipitate to dissolve it. Then, physiological saline is added and methanol is added again. After stirring, the methylene chloride solution is separated and the aqueous layer is removed.

9. Dewater the separated methylene chloride solution and store the obtained lipid mixture at low temperature.

The composition of the lipid mixture obtained by this method is 25 to 30% of cerebroside, 16 to 35% of phosphatidylethanolamine, 16 to 24% of cerebroside sulfate, 8 to 12% of phosphatidylcholine, 11 to 20% of phosphatidylserine, and 4-6% sphingomyelin. IC ₅₀ of the composition based on this composition (concentration with 50% antihemolytic) is 4-10 microgram / ml.

Anti-hemolytic compositions are prepared in the form of liposomes smaller than 1 μm to increase the total surface area as part of intravenous injection and to increase activity.

The method for this is described as follows. The hemolytic composition is obtained by dissolving a lipid mixture including SL in a chloroform, methanol, and ethanol solvent in a nitrogen atmosphere, and then distilling the solvent under reduced pressure and adding saline solution. Fine particles are produced by uniformly dispersing the particles under ultrasonic pressure and high pressure. Sterile suspensions are prepared by passing a filter from 0.7 to 0.4 μm under aseptic conditions.

The present invention provides compositions useful for the prevention and inhibition of hemolysis due to other etiologies and in particular complement induced hemolysis.

A. Composition for inhibition of complement-induced hemolysis

The present invention suggests that SL inhibits complement-induced hemolysis. This type of hemolysis occurs in hemolytic diseases of the newborn and diseases such as paroxysmal nocturnal hemoglobinuria, and complement-induced hemolysis is caused by the action of drugs and infection by microorganisms. [F.E. Bercovitz (1991) Rev. Infect. Dis V 13, pp 1151-1162.

Compositions containing SL inhibit complement-induced hemolysis from concentrations of 0.0005 mg / ml to 20 mg / ml. SL accounts for at least 10% of all polar lipids. Polar lipids are natural products such as cerebroside, cholesterol, diphosphatidylglycerol, phosphatidic acid, phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, phosphatidyl inositol, phosphatidylserine, sphingomyelin and include chemical synthetic derivatives of the polar lipids . Preferred compositions of the present invention are when it contains sulfolipid (10-100%), phosphatidylcholine (0-90%), cerebroside (0-40%), cholesterol (0-60%).

Other polar lipids may also be included in the present compositions but may lower antihemolytic activity. When blood transfusion is always followed by undesired blood hemolysis, the anti-hemolytic composition of the present invention can be used in addition to the blood, in which case it is applied as a composition such as treatment for complement-induced hemolysis. At this time, the concentration of the composition containing a preferred SL is 0.005mg / ml ~ 20gm / ml.

B. Nasal blood compositions (inhibition of hemolysis by agents such as surfactants and antibiotics)

The present invention provides a nasal blood composition suitable for intravenous injection of mammals including humans by incorporating a pharmacologically active but inducing blood hemolysis like SL. In addition to immune hemolysis, it is important to find substances that have a strong affinity for substances that cause the destruction of red blood cells for inhibition of non-immune hemolysis.

Since the anti-hemolytic composition of the present invention has a strong negative charge, it is possible to reduce hemolysis due to a substance having a strong positive charge or dissolved in a lipid duplex. Sephacrochlor is a cephalosporin-based antibiotic known to cause hemolysis. The anti-hemolytic composition of the present invention inhibits hemolysis by this agent. The effective concentration in this case is higher than that of complement-induced hemolysis, from 0.02 mg / ml to 20 mg / ml. The composition is sulfolipid (5-100%), phosphatidylcholine (0-95%), cholesterol (0-60%), cerebroside (0-50%), phosphatidylethanolamine (0-30%), Phosphatidylserine (0-30%) and sphingomyelin (0-35%). In the case of the lauryl sulfite surfactant, hemolysis can be induced, and hemolysis can be suppressed by adding the anti-hemolysis composition of the present invention having the above composition.

C. Composition for inhibition of hypotonic hemolysis

Animal cells break through the cell membrane in the reservoir due to the difference in the osmotic pressure inside and outside the cell. Hypolytic hemolysis is also induced by some infections. (M. L. Bennish et al (1990) J. Infect Dis. V. 161. pp 500-506).

Plasma protects hypotonic hemolysis of red blood cells. Plasma constituents that protect against hemolysis surround the cell membranes of red blood cells exposed to the stock solution, and have a strong negative charge to combine with sodium ions to form an osmotic active layer to prevent excess water from entering the cell. (T. Butler, CA Bradley, JE Owensby (1992) Int. J. Exp. Pathol, V. 73.p 2733).

SL in the present invention is a strong acidic group has a negative charge and the anti-hemolytic composition of the present invention has a protective function against hypotonic hemolysis. In this case, the effective concentration is 0.01mg / ml ~ 0.02mg / ml and the optimal composition is sulfolipid (16 ~ 100%), phosphatidylcholine (0 ~ 84%), cholesterol (0 ~ 60%), cerebroside (0 ~ 50%).

In the treatment of diseases involving complement-induced hemolysis, including red blood cell hemolysis of the newborn, paroxysmal nocturnal hemoglobinuria, etc., the concentration of the anti-hemolytic composition of the present invention is 14-30 mg / kg in isotonic solution and intravenous injection. Is administered. In the case of blood transfusion, 2 ~ 5% suspension with isotonic solution is administered to the blood at a concentration of 0.005mg / ml ~ 5mg / ml. In the use of a drug that induces hemolysis, the anti-hemolytic composition of the present invention is administered at 14 to 300 mg / kg by intravenous injection.

(Production Example)

This preparation example shows a method for preparing the anti-hemolytic composition of the present invention from bovine spinal cord. The method is also suitable for extracting sulfolipids contained in spinal cord, brain or tissue cells of other animals. All operations are carried out under a stream of nitrogen.

Stage 1.Preparation of Lipid Mixture Containing Sulpholipids

1 kg of frozen bovine spinal cord is ground uniformly and 0.5-1 L of acetone is added. The ground small water is introduced into the reactor and cooled acetone at -10 ° C is added. The mixture is stirred for 5 minutes and filtered through a Nutsch filter. The acetone solution is removed and the residue is transferred to the reactor. Acetone cooled to −10 ° C. is added and stirred vigorously for 2 hours. Filter the residue with a Nash filter (acetone extract 2.) Repeat this operation once more. (Acetone extract 3). The residue is washed with 0.2 L methanol in a Nash filter. Acetone extracts 2 and 3 are used for cholesterol isolation. Put the residue in the reactor. Methanol is added. The mixture is stirred at 20-22 ° C. for 2 hours. The residue is filtered through a Nash filter.

Methanol extract (Extract 1) is placed in the refrigerator. Put the residue in the reactor. Hexane is added and stirred for 2 hours. The residue is filtered through a Nash filter. Hexane extract (extract 2) is placed in the refrigerator. Put the residue in the reactor. Hexane is added. The mixture is stirred vigorously for 2 hours and the residue is filtered through a nut filter. Hexane extract (extract 3) is placed in the refrigerator. Extracts 1,2 and 3 are evaporated to dryness in vacuo at 35-40 ° C.

The residue is taken up in 0.5 L methyl chloride. 2.5 L acetone is added to the aqueous lipid solution with stirring. The mixture is placed in a cold box (0-4 ° C.) for 18 hours, and the supernatant is removed after centrifugation of the precipitate. The precipitate is dissolved in 0.3 L methyl chloride. Acetone is added to the lipid solution and the mixture is placed in the cold box for 18 hours. Centrifuge the load and remove the supernatant. The precipitate is dissolved in 0.3 L methyl chloride. Methanol is added to the lipid solution with stirring and the mixture is evaporated. The mixture is placed in a cold box for 18 hours, centrifuged and the supernatant is removed. The precipitate is dissolved in 0.3 L methyl chloride and 1.5 L methanol is added to the lipid solution. The mixture is placed in a cold box for 18 hours and the precipitate is centrifuged and the supernatant removed. The precipitate is dissolved in 0.3 L methyl chloride. 0.3 L physiological saline and 0.1 L methanol are added to the lipid solution and the mixture is stirred for 5 minutes.

The methyl chloride solution is separated through a separator and the aqueous layer is removed. The methyl chloride solution is dehydrated with 0.1-0.2 kg Na ₂ SO ₄ and placed in a cold box for 2 hours. The methyl chloride solution is filtered off and Na ₂ SO ₄ is removed. Put methyl chloride lipid solution in -10 ~ -20 ℃ refrigerator and store. The concentration of lipids is determined.

Stage 2. Preparation of Anti-Hemolytic Composition of the Present Invention

The methyl chloride solution is evaporated and dispersed in physiological salts, and then the concentration of lipid is dispersed to 2.6%. Dispersion of liposomes is performed three or four times through a microfluidizer (up to the same optical diversity) and the dispersion is sterilized. The dispersion is filtered through a 0.4 μm filter and the concentration of lipids is determined. The dispersion is diluted in 2% sterile isotonic solution and the prepared lipids are inserted into sterile bottles.

(Example)

The activity of anti-hemolysis was measured by the following method.

Reagent 1: Add 1 ml of red blood cells to 9 ml of saline solution. Mix well for 30 seconds, then centrifuge to discard supernatant. Repeat this process two or three times. 0.3 ml of the red blood cells thus obtained are mixed with 9.7 ml of saline solution.

Reagent 2: Dilute 0.1 ml of hemolytic serum to 30 ml of saline solution (the concentration of erythrocyte hemolysis for 20-45 minutes at 35 degrees Celsius and the volume ratio of diluted serum and erythrocytes shall be 1: 1).

Reagent 3: 8 mg of complement was dissolved in 1 ml of saline, and diluted again with 9 ml of saline.

(reaction)

1. Main Control: The reagents were mixed in the following order.

0.5 ml of reagent 1 + 0.5 ml of reagent 2

Reagent 3 0.5ml + Saline 1ml

2. Red blood cell control: The reagents were mixed in the following order.

Reagent 1 0.5ml + Saline 2ml

3. liposome dispersion control; The reagents were mixed in the following order.

0.5ml of anti-blood composition + 2ml of saline solution

4. Main experiment: The reagents were mixed in the following order.

0.5 ml of reagent 1 + 0.5 ml of anti-blood composition + Reagent 2

0.5ml + Reagent 3 0.5ml + Saline 0.5ml

These samples were incubated and centrifuged at 30 degrees Celsius until complete hemolysis of the major controls occurred. The supernatant was diluted 8-fold and its absorbance was measured at 418 nm. Antihemolytic activity was calculated according to the following formula.

AA = [(ODcont-(ODex-ODlS)) / ODcont] * 100% Formula (4)

Where ODex is the absorbance of the experiment at 418 nm, ODcont is the major large group of absorbance at 418 nm, and OSls represents the absorbance for the lipid suspension at the same concentration as in the main experiment. Thus, the anti-hemolytic activity was measured and then the concentration showing the activity of IC _50, i.

(Examples 1-16)

The following example shows the anti-hemolytic activity of the complex comprising SL and SL.

Table 1 IC ₅₀ for Formulations Including SL

Example 1 and Example 2 show that each serebroside sulfite (CS) purified from bovine brain and spinal cord exhibits substantially the same antihemolytic activity. Examples 3-6 show that in two compositions, egg phosphatidylcholine (PC) increases antihemolytic activity, while sphingomyelin (SM) decreases antihemolytic activity. Example 7 and Example 8 show anti-hemolytic activity of sulfolactosyl ceramide (SLC) and sulfogalactosyldiacylglycerol (SGDAG). Examples 9 to 13 showed the anti-hemolytic activity of the three compositions, the activity was increased in the case of the addition of cerebrose (Cer) and cholesterol (Chol) in this composition. In Example 14, it can be seen that the four compositions of cerebromide and cholesterol mixed together increase the anti-hemolytic activity of the cerebromide sulphite. Example 15 shows the strong haemolytic activity of the composition isolated from the spinal cord of the bovine prepared in the preparation example. Example 16 shows the difference in anti-hemolytic activity in liposomes and lipid suspensions.

(Examples 17-20)

In Examples 17 to 20, immunohemolysis was suppressed by the anti-hemolysis composition of the present invention in hemolysis of newborns. The experimental process is as follows.

Erythrocytes of hemolytic newborns and serum of their mothers were used. Remove the fibrin in the blood drawn from the cleansing vein of the neonatal fronthead, add 9 ml of physiological saline to 1 ml of the red blood cells, mix well for 30 seconds, and discard the supernatant by centrifugation. Repeat this process twice and add 9.7 ml of saline solution to 0.3 ml of red blood cells. Blood drawn from the mother of the newborn is left for 20 hours at 20 degrees Celsius and then centrifuged. The obtained serum was diluted 10 times with physiological saline. In the main experiment, 0.5 ml of red blood cells were added 0.5 ml of anti-hemolytic composition of the present invention, 0.5 ml of serum, and 0.5 ml of physiological saline. In a blank experiment, 0.5 ml of red blood cells and 0.5 ml of serum and 1 ml of physiological saline are added. 1 ml of serum 0.5 ml physiological saline is added to 0.6 ml of the anti-hemolytic composition of the present invention in the light scattering control group. All samples were measured at 418 nm and the degree of hemolysis was calculated by the following formula.

H = (ODex -ODls-ODbl) * 100 / OD ₁₀₀ Formula (5)

Where ODex is the absorbance in the main experiment, ODbl is the absorbance in the blank experiment, OD ₁₀₀ is the absorbance under 100% hemolysis, and ODls is the absorbance for the lipid suspension at the same concentration as in the main experiment. This experiment was carried out by setting the final concentration of the anti-hemolytic composition of the present invention to 0.04, 0.2, 1 mg / ml. The results are shown in Table 2.

Table 2 Hemolysis degree by concentration of anti-hemolytic composition of the present invention (%)

As shown in the table, in the absence of the anti-hemolytic composition of the present invention, hemolysis was 21-48% according to the degree of disease, and in all cases, significant delay in hemolysis occurred at a concentration of 0.2 mg / ml or more.

(Example 21)

This example shows the inhibition of complement induced hemolysis by the antihemolytic composition of the present invention in the case of paroxysmal nocturnal hemoglobinuria. The experimental process is as follows.

Blood from patients with paroxysmal nocturnal hemoglobinosis and normal blood were collected as controls and sodium oxalate or sodium citrate was added as anticoagulant. The composition of the hemolysis system was mixed according to Table 3 and the anti-hemolysis composition of the present invention was added to test tube No. 2. This test tube was incubated for 1 hour at 37 degrees Celsius. Mix supernatant with 4.7 ml of 0.04% ammonium hydroxide. In a blank experiment, 4.7 ml of 0.04% ammonium hydroxide is mixed with 0.3 ml of serum. 4.7% of 0.04% ammonium hydroxide was added to 0.025ml of red blood cells from the patient and 0.275ml of saline solution was added to perform 100% hemolysis. All samples were measured for absorbance at 418 nm and the degree of hemolysis was calculated according to the following formula.

H = (ODex-ODls-ODbl) * 100 / OD ₁₀₀ General formula (5)

Where ODex is the absorbance in the main experiment, ODbl is the absorbance in the blank experiment, OD ₁₀₀ is the absorbance under 100% hemolysis, and ODls is the absorbance for the lipid suspension at the same concentration as in the main experiment.

TABLE 3 Anti-hemolytic activity of anti-hemolytic compositions of the present invention against paroxysmal nocturnal heomglobinuria

(Examples 22-29)

This example describes the treatment of anti-hemolytic and complement-induced hemolysis and associated disorders and diseases by the anti-hemolytic composition of the present invention for immune hemolysis in sheep and rabbits in mammals.

TABLE 4 Inhibition of Complement Induced Hemolysis in Animals and Anti-hemolytic Activity of Anti-Hemolytic Composition of the Present Invention

(Examples 30-41)

This example illustrates the antihemolytic properties of the antihemolytic composition of the present invention in the case of hemolysis due to Staphylococcus toxin, venom toxin, insect toxin. It is well known that toxins in snakes and insects have a hemolytic effect. An important factor causing the hemolytic response among the components of these toxins is phospholipase.

Phospholipases break down the ester bonds of phospholipids to form lyso-phospholipids, which break through the lipid bilayer of the cell membrane. Phospholipase C is produced in microorganisms and has a hemolytic effect. In this case diacyldiglycerides are formed and this kind of lipid has a very small polar head.

Thus, they fail to form lipid bilayers and destroy cell membranes. Liposomes containing SLs that have strong negative charges against substances that destabilize these cell membranes are good targets of cell membrane dependent proteins such as phospholipases. Thus, liposomes prevent, inhibit and inhibit hemolysis.

Experimental procedure: Add 1 ml of red blood cells to 9 ml of saline solution. Mix well for 30 seconds, then centrifuge to discard supernatant. Repeat this process two or three times. 0.3 ml of the red blood cells thus obtained are mixed with 9.7 ml of saline solution. Insect venom was diluted in physiological saline to 0.15 mg / ml, phospholipase C was diluted to physiological saline to 10 mg / ml, and alpha-hemolysin was used commercially without dilution. To 0.25 ml red blood cells, 0.25 ml of the anti-hemolytic composition (20 mg / ml) of the present invention was added, followed by 0.05 ml of toxin. As a control experiment, an equal volume of physiological saline was added instead of the anti-hemolytic composition of the present invention.

These samples were incubated and centrifuged at 30 degrees Celsius until complete hemolysis of the major controls occurred. The supernatant was diluted 8-fold and its absorbance was measured at 418 nm. Antihemolytic activity was calculated according to the following formula.

AA = [(ODcont- (ODex-ODls)) / ODcont] * 100% Formula (4)

Where ODex is the absorbance of the main experiment at 418 nm, ODcont is the absorbance of the main control at 418 nm, and ODls represents the absorbance of the lipid suspension at the same concentration as in the main experiment.

TABLE 5 Inhibition of hemolysis due to toxin containing phospholipase A ₂ and phospholipase C using the anti-hemolytic composition of the present invention

The antihemolytic compositions of the present invention containing stearylamine to reduce negative charges in Examples 31,34,37,40 reduce antihemolytic activity. This confirms the importance of the components of the antihemolytic composition of the present invention having a charge.

(Examples 42 to 45)

This example tested the antihemolytic properties of the haemolytic composition of the present invention against Sephacro, a cephalosporin antibiotic with a hemolytic effect.

Experimental process

1) Determination of saturation concentration of Sephacrochlor: 5mg / ml

2) Determination of Sephacrore Concentration causing Hemolysis

0.25ml erythrocyte + 0.9ml saline solution + 0.1ml sephacro

2.0.25ml red blood cell + 0.8ml saline solution + 0.2ml sephacro

3.0.25ml Red Blood Cell + 0.7ml Saline + 0.3ml Sephacro

4.0.2ml red blood cells + 0.5ml saline + 0.5ml Sephacro

After incubation for 1 hour and 30 minutes at 37 degrees Celsius, hemolysis was measured. Only about 4% of hemolysis was observed in sample 4, and sample 4 was applied to the main experiment.

3) Main experiment

0.25ml erythrocyte + 0.25ml 1.0% antihemolytic composition of the present invention + 0.5ml Sephacro + 0.25ml physiological saline

2. 0.25ml erythrocyte + 0.25ml 1.0% antihemolytic composition of the present invention + 0.5ml Sephacro + 0.25ml physiological saline

3. 0.25ml erythrocyte + 0.25ml 1.0% antihemolytic composition of the present invention + 0.5ml Sephacro + 0.25ml physiological saline

At the same time as the main control was added the same volume of physiological saline instead of the anti-hemolytic composition of the present invention, and cultured for 1 hour and 30 minutes at 37 degrees Celsius, the supernatant was diluted 8-fold and the absorbance at 418 nm was measured.

TABLE 6 Anti-hemolytic properties of anti-hemolytic compositions of the present invention against hemolysis caused by Sephacrocro

(Examples 46-50)

This example shows the anti-hemolytic activity of the anti-hemolytic composition of the present invention in the case of hemolysis with a surfactant including lauryl sulfite. A concentration of 0.004 mg / ml of laurylsulphite was used, which resulted in complete hemolysis in 10 minutes.

Experimental process

1) Determination of concentration of lorylsulfite causing hemolysis

1.0.25ml Red Blood Cell + 0.25ml Saline + 0.25ml 0.5% Lorylsulfite

2.0.25ml Red Blood Cell + 0.25ml Saline + 0.25ml 0.05% Laurel Sulfit

Red blood cell + 0.25ml physiological saline + 0.25ml 0.025% L'oryl sulfite

Red blood cell + 0.25ml physiological saline + 0.25ml 0.0125% L'oryl sulfite

Erythrocyte + 0.25ml physiological saline + 0.25ml 0.00625% Lorylsulfite

Incubated for 5 minutes at 20 degrees Celsius and the degree of hemolysis was calculated. Hemolysis completely occurred in sample 4 and 20% hemolysis occurred in sample 5. Thus sample 4 was selected.

2) Main experiment

0.25ml erythrocyte + 0.25ml 1.0% antihemolytic composition of the present invention + 0.25ml 0.125mg / ml laurylsulphite

2. 0.25ml erythrocyte + 0.25ml 1.0% antihemolytic composition of the present invention + 0.25ml 0.125mg / ml laurylsulphite

3. 0.25ml erythrocyte + 0.25ml 1.0% antihemolytic composition of the present invention + 0.25ml 0.125mg / ml laurylsulphite

4. 0.25ml erythrocyte + 0.25ml 1.0% antihemolytic composition of the present invention + 0.25ml 0.125mg / ml laurylsulphite

5. 0.25ml erythrocyte + 0.25ml 1.0% antihemolytic composition of the present invention + 0.25ml 0.125mg / ml laurylsulphite

At the same time as the main control was added the same volume of physiological saline instead of the anti-hemolytic composition of the present invention and incubated for 20 minutes at 20 degrees Celsius and centrifuged to dilute the supernatant 8 times and then measured the absorbance at 418 nm.

TABLE 7 Anti-hemolytic properties of anti-hemolytic compositions of the present invention against hemolysis caused by lorylsulfite

(Examples 51 to 56)

In the present embodiment relates to the inhibition of hemolysis due to the stock solution.

Experimental process

1) Determination of Sodium Chloride Concentration in Hemolysis

0.25 ml red blood cells + 0.75 ml 0.81% sodium chloride

2.0.25 ml red blood cells + 0.75 ml 0.76% sodium chloride

3.0.25 ml red blood cells + 0.75 ml 0.63% sodium chloride

4.0.25 ml red blood cells + 0.75 ml 0.54% sodium chloride

5.0.25 ml red blood cells + 0.75 ml 0.45% sodium chloride

6.0.25 ml red blood cells + 0.75 ml 0.36% sodium chloride

Hemolysis occurred immediately after the addition of sodium chloride urine in Samples 5 and 6, 45% of solution in Sample 2 and 85% in Sample 3 after incubation at 20 ° C for 15 minutes.

2) Main experiment

1.0.2 ml red blood cells + 0.25 ml 0.01% antihemolytic composition of the present invention + 0.75 ml 0.63% sodium chloride

2. 0.25 ml red blood cells + 0.25 ml 0.02% antihemolytic composition of the present invention + 0.75 ml 0.63% sodium chloride

3. 0.25ml red blood cells + 0.25ml 0.01% antihemolytic composition of the present invention + 0.75ml 0.76% sodium chloride

4. 0.25ml erythrocytes + 0.25ml 0.02% antihemolytic composition of the invention + 0.75ml 0.76% sodium chloride

At the same time as the main control was added the same volume of sally saline instead of the anti-hemolysis composition of the present invention, 20 minutes at 20 degrees Celsius and centrifuged to dilute the supernatant 8-fold, the absorbance was measured at 418 nm.

TABLE 8 Anti-hemolytic properties of anti-hemolytic compositions of the present invention against hemolysis caused by stock solutions

Claims (6)

After grinding fresh bovine or mammalian furnace or spinal cord, adding acetone, removing acetone soluble substance and acetone solution, collecting methanol extracted by filtration with methanol, and extracting hexane solution by adding hexane, The collected extract was distilled under reduced pressure, methylene chloride was added to dissolve, acetone was added, methylene chloride was added to the precipitate, dissolved, and methanol was added. The mixture was distilled under reduced pressure to 1/3 volume, left to precipitate, and methylene chloride was added thereto. After dissolving, adding physiological saline solution and methanol again, the methylene chloride solution is separated and the aqueous layer is removed, followed by dehydration of the separated methylene chloride solution. The composition of the haemolytic lipid composition according to claim 1, wherein the composition of the haemolytic lipid composition is 25% to 30% of cerebroside, 16% to 25% of phosphatidylethanolamine, 16% to 24% of cerebroside sulfate, 8% to 12% of phosphatidylcholine, and 11% to 20% of phosphatidylserine And 4 to 6% sphingomyelin. Antihemolytic agent consisting of a lipid mixture comprising sulfolipids with a negative charge of formula (I) LR-SO ₃ X (I) In the above formula L is the lipid moiety (the lipid moiety has a sterol structure such as two hydrocarbon chains or cholesterol); R is a carbohydrate moiety (selected from the group consisting of glucose, galactose, mannose, ribose, 2-deoxyribose, and combinations of two or more thereof); X is hydrogen or cation (Li, Na, K, 1 / 2Mg, 1 / 2Ca) contained in normal blood. 4. The anti-hemolytic agent according to claim 3, wherein the negatively charged sulfolipid is cerebroside-3-sulfate, sulfolactosyl ceramide, sulfogalactosyl diacylglycerol or sulfogalactosylalkylacyl glycerol. 4. The anti-hemolytic agent according to claim 3, wherein the content of sulfolipid of formula (I) in the lipid mixture comprises 16 to 90% of the total polar lipid content. The composition according to any one of claims 3 to 5, wherein the lipid mixture is composed of sulfolipid 16-90%, phosphatidylcholine 10-74%, cerebroside 10-40%, cholesterol 0-50%. Hemolytic