JP2003055236A - Eosinophilic active substance, method for producing the same and pharmaceutical preparation - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new eosinophilic active substance from a deep ocean water. SOLUTION: The eosinophilic active substance is a component of a polar solvent soluble fraction transferred from a solute of a deep ocean water to a polar solvent phase by contacting the solute of deep ocean water separated from deep ocean water with the polar solvent. The eosinophilic active substance can be produced in high concentration by the steps of separating a substance dissolved in deep ocean water from the water, contacting the separated solute of the deep ocean water with a polar solvent to transfer a polar solvent-soluble substance in the deep ocean water solute to the polar solvent phase, separating the product into the polar solvent phase and the component insoluble in the polar solvent and removing the polar solvent from the separated polar solvent phase to obtain the eosinophilic active substance existing in the polar solvent soluble component. The eosinophilic active substance is effective for the treatment of atopic dermatitis, etc.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an eosinophil active substance contained in deep sea water, a method for obtaining a high concentration of the eosinophil active substance from deep sea water, and a drug containing the eosinophil active substance. Regarding

[0002]

TECHNICAL BACKGROUND OF THE INVENTION Allergies such as atopy are a type of hypersensitivity in which a genetic constitution plays a major role. The antigen that causes such allergic symptoms is believed to be in the blood, and the complex of this antigen and antibody releases histamine or histamine-like substance from the tissue, resulting in specific symptoms. It is believed that. For example, in atopic dermatitis, peripheral blood eosinophils are increased and histological findings often show eosinophil infiltration. From this, it is considered that eosinophils are one of the cells that play a central role in atopic dermatitis.

Against this background, various studies have been conducted on active substances for eosinophils, and it is already known that deep sea water enhances the activity of eosinophils. Deep-sea water is seawater with a high specific gravity that flows very slowly over the deep-sea floor with a depth of 200 m or more. It flows through the deep-sea floor where sunlight does not reach, and because it has a high specific gravity, there is absolutely no ocean current flowing through the surface layer of the sea. It is thought to have a different composition or properties from the seawater in the surface layer because it slowly flows through the seabed through different routes and is not mixed with the seawater in the surface layer.

As described above, the existence of deep sea water was generally recognized only recently, and until then, little was known about the details of such deep sea water. However, after the existence of such deep sea water became known, various proposals using such deep sea water have been made, for example, containing rare minerals which are abundantly contained in deep sea water. Drinking water has also been sold.

[0005] Apart from the purpose of deep sea water aimed at such commercial effect, the components contained in the deep sea water are analyzed, and the relationship between the components contained in these deep sea water and the medicinal effect is examined. Attempts have also been made to use deep sea water as a therapeutic agent for various diseases. Since deep sea water is seawater with a depth of 200 m or more that does not reach the light, unlike surface water, photosynthesis by phytoplankton or seaweed is not carried out, and nitrogen, phosphorus, consumed by photosynthesis,
Silicon, nitric acid, etc. are contained at a higher concentration than surface water, and since photosynthesis is not carried out, it is considered that there are less organic substances and less pathogenic bacteria than surface water. For this reason, conventional applications of deep sea water have been mainly based on inorganic substances abundantly contained in deep sea water.

[0006] That is, conventionally, as the deep sea water, a separated product (deep water separated product) from the deep water obtained through the concentration step, the desalting step and the separation step has been used. The water separation product contains a large amount of inorganic substances. That is, the infrared absorption spectrum of the substance obtained through the concentration step, the desalting step and the separation step has a peak very similar to the infrared absorption spectrum of calcium sulfate. It is known that calcium sulfate has substantially no medicinal effect related to allergy. Therefore, in the conventional antiallergic drug using deep sea water, the deep water isolate compounded as its medicinal component is calcium sulfate, which is hardly related to the antiallergic drug effect. If it is possible to separate the components exhibiting antiallergic drug efficacy from, the antiallergic drug having higher efficacy, especially against skin diseases such as atopic dermatitis, which are recently increasing with changes in the environment, etc. It becomes possible to provide a highly useful drug.

[0007]

It is an object of the present invention to provide an isolate containing a high concentration of a component having a high eosinophil activity, which is effective against atopic dermatitis, etc. from deep sea water. More specifically, the present invention provides a method for efficiently obtaining an isolate containing a high concentration of a component that alleviates allergic symptoms from deep sea water, and a component containing a high concentration of an eosinophil active substance obtained by this method. It is intended to be provided.

Another object of the present invention is to provide a drug containing the eosinophil active substance in a high concentration.

[0009]

SUMMARY OF THE INVENTION The present invention provides eosinophils contained in a polar solvent-soluble component that is transferred from a deep aqueous solution separated from deep sea water to a polar solvent by contact with the polar solvent. In active substances. Further, the present invention is a step of separating a substance dissolved in deep sea water from the deep sea water, a polar solvent contained in the deep sea water by contacting the separated deep sea water present with a polar solvent. A step of transferring a soluble substance to a polar solvent phase, a step of separating the polar solvent phase and a component insoluble in the polar solvent, and a polar solvent-soluble component by removing the polar solvent from the separated polar solvent phase. The method for obtaining an eosinophil active substance at a high concentration is characterized by including a step of obtaining the eosinophil active substance contained in.

Further, the present invention relates to eosinophils contained in a polar solvent-soluble component which is transferred from the deep aqueous solution to the polar solvent phase by contact between the deep aqueous solution separated from the deep sea water and the polar solvent. It is a pharmaceutical product characterized by containing an active substance. In the present invention, it is desirable that the polar solvent is a polar solvent having a dielectric constant within a range of 2.0 to 200, preferably 20 to 81.0, and the deep ocean water is collected from a deep sea at a depth of 200 m or more. It is preferable that the deep seawater separated from the deep seawater is concentrated to 1/5 to 1/1000 of the initial amount of the deep seawater, and further desalted to thereby concentrate the deep seawater. It is preferably a precipitate from water.

This eosinophil active substance is usually transferred to the polar solvent phase obtained after contact with the infrared absorption spectrum (1) measured for deep-layer aqueous substances separated from deep sea water and the polar solvent. By comparing with the infrared absorption spectrum (2) measured for the component that was not present,
It is a substance characterized by a peak that is present in the infrared absorption spectrum (1) but not in the infrared absorption spectrum (2).

In the method of the present invention, the separated deep-layer water-soluble substances are sequentially contacted with a plurality of polar solvents having different dielectric constants, and the polar solvent-soluble components contained in the deep-layer water-soluble substances are sequentially added. It is preferable to transfer to a polar solvent layer and separate. By operating as described above, the component soluble in the polar solvent can be efficiently separated from the calcium sulfate from the deep seawater concentrated and desalted. The components extracted into the polar solvent here are considered to be mainly organic substances, and the components separated in this way show higher eosinophil activity.

[0013]

DETAILED DESCRIPTION OF THE INVENTION Next, the eosinophil-active substance of the present invention,
A method for obtaining a high content ratio of this eosinophil active substance from deep sea water, and a drug containing this eosinophil active substance will be specifically described with reference to the process diagram illustrated in FIG. 1. As shown in FIG. 1, the eosinophil-active substance of the present invention is obtained by using deep sea water as a raw material.

Generally, the depth to which sunlight can reach is approximately 200 m in ocean water, and in the present invention, deep ocean water having a depth of 200 m or more, preferably 300 m or more, which sunlight does not reach is used. Although the water depth is less than 200m, deep sea water that rises near the sea surface due to the upwelling of seawater can also be used. Since sunlight naturally does not reach deep ocean water that exists in the deep ocean, photosynthesis by algae or aquatic plants does not occur in this deep ocean water, and there is almost no breeding of plankton. The general idea is not to. Therefore, the deep ocean currents contain almost no organic substances, but on the other hand, they contain minerals at a higher concentration than ordinary ocean water (meaning ocean water other than deep ocean water). The minerals also contain rare minerals that tend to be deficient in the current diet. Therefore, in the conventional deep sea water, it has been considered that the above-mentioned rare minerals act in a complex manner and have activity on eosinophils.

However, when the present inventor analyzed the deep sea water in detail, it was found that surprisingly many organic substances were dissolved in the deep sea water. Then, a trace amount of organic substances contained in the deep sea water can be relatively easily separated from the deep sea water by extraction using a polar solvent. If such a component can be efficiently extracted with a polar solvent and then the extraction solvent can be removed, a substance having high eosinophil activity can be taken out, and such an eosinophil active substance is allergenic such as atopy. Very useful as a therapeutic drug for diseases.

Hereinafter, the eosinophil-active substance of the present invention will be described in more detail along the manufacturing method shown in the process chart of FIG. In the present invention, the deep sea water used to obtain the eosinophil active substance has a water depth of 200 m as shown in FIG.
Or more, preferably 300 m or more, more preferably 300 to 70
Deep sea water collected from a deep sea of 0 m or more is usually used after being filtered in order to remove suspended solids that may be mixed in at the time of collection. The opening is usually 0.1 for this filtration.
Use a filter (A) of about 0.5 μm. By filtration using this filter (A), a seawater suspension that may be contained in deep sea water and a deep sea water (filtrate) are separated.

The seawater suspension obtained by separating the deep seawater obtained as the filtrate as described above is discarded in the present invention, and the deep seawater obtained as the filtrate by the filtration is used in the present invention. . Then, the deep sea water obtained as this filtrate is usually 1/5 to 1/1000, preferably 1/1 in volume ratio with respect to the deep sea water used first.
Concentrate to an amount of 0 to 1/100. The concentration is usually 0 to 100 ° C., preferably 10 to 40 ° C. under reduced pressure of 0.1 to 100 Torr, preferably 1 to 20 Torr.
Performed at the temperature of. FIG. 1 shows a mode in which deep ocean water is distilled and concentrated to 1/10 at a temperature of 40 ° C. or lower under a reduced pressure condition of 20 Torr. By thus concentrating the deep sea water at a low temperature under reduced pressure, it is possible to prevent the organic compound contained in the deep sea water from being decomposed or reacted by a concentrating operation such as heat.

By concentrating the deep sea water in this manner, a part of the components dissolved in the deep sea water is precipitated. The components thus deposited are filtered (B)
Separate with. As the filter (B), a filter having an opening of 0.1 to 5 μm, preferably about 0.2 to 0.5 μm, such as a membrane filter, can be used.

The solid matter (precipitation residue part (1)) separated by the filter (B) is used in the subsequent step. On the other hand, the filtrate obtained by filtration using the filter (B) is concentrated and desalted at least once, preferably twice or more, and then desalted and concentrated. For concentration, the final amount of the liquid is usually 1/5 in volume ratio with respect to the amount of the filtrate before the concentration.
Concentrate to ~ 1/1000, preferably 1/10 to 1/100. This concentration is usually 0.1-100
Torr, preferably 1-20 Torr under reduced pressure, usually 0-
It is carried out at a temperature of 100 ° C, preferably 10-40 ° C.
FIG. 1 shows a mode in which distillation and concentration are performed at a temperature of 40 ° C. or lower under a reduced pressure condition of 20 Torr. The liquid thus concentrated is desalted using a desalting device such as a desalting device such as a mosaic charged membrane or a semipermeable membrane. Finally, 70% by weight or more, preferably 90% by weight or more, of chlorine-based salts contained in the filtrate finally obtained by filtration with the filter (B) is removed.

Since the solid content (precipitation residue part (2)) is precipitated by performing the above-mentioned concentration and desalting at least once, preferably twice or more, particularly preferably 5 to 10 times,
The solid content is filtered using a filter (C). Here, the filter (C) is usually 0.1 to 5 openings.
It is possible to use a filter of μm, preferably 0.1 to 0.5 μm, such as a membrane filter.

In the present invention, this filtered solid content (precipitation part residue (2)) is used. Therefore, if the filtrate obtained by the above filtration has no activity as a result of eosinophil activity test confirmation, It is abandoned in the invention. The solid content (precipitation part residue (2)) obtained as described above is mixed with the solid matter (precipitation part residue (1)) collected in the previous step to give the eosinophil-active substance of the present invention. Used to get. In the usual case, when, for example, 20 liters of deep sea water was first used, a mixture of this solid matter (precipitation residue (1)) and solid matter (precipitation residue (2)) (separated from deep ocean water) The total of deep water soluble components) is about 5 to 50 g, preferably 10 to 4 g.
It is about 0 g, and the amount of the deep water-soluble component obtained based on the weight of the deep sea water initially used at this stage is usually 0.
It is 025 to 0.25%, preferably 0.05 to 0.2% by weight. FIG. 2 shows a chart of the infrared absorption spectrum (1) of the deep water-soluble component separated from the deep sea water.

Further, since the deep water-soluble component separated from the deep sea water exhibits characteristics relatively similar to that of calcium sulfate in appearance, the infrared absorption spectrum of a special grade reagent of calcium sulfate was measured as a reference, and this infrared ray was detected. An absorption spectrum (3) chart is shown in FIG. Comparing Fig. 2 and Fig. 3, the deep water soluble components separated from the deep sea water were 871 cm ^-1 , 1406 cm ^-1 , 1477 cm.
^{-1, 1540cm -1, 2113cm -1,} etc. in the vicinity of 2974cm ^-1, it can be seen that a peak not found in calcium sulphate is guaranteed reagent. That is, the deep-water-soluble component separated from the deep-sea water contains a relatively large amount of calcium sulfate, but the calcium sulfate contains a component having the infrared absorption spectrum as described above. it is conceivable that.

Therefore, in the present invention, in order to separate the deep-water-soluble components separated from the deep sea water obtained as described above from calcium sulfate, these components are extracted and separated using a polar solvent. As the polar solvent, one having a dielectric constant of 2 to 200, preferably 20 to 80 is used. The dielectric constant of these polar solvents may be a value of a single polar solvent or a composite value when a plurality of polar solvents are mixed and used.

Examples of such polar solvents include chloroform (dielectric constant: 4.806) and carbon tetrachloride (dielectric constant:
2.24) and other chlorine-based / halogen-based organic solvents, methanol (dielectric constant: 32.65), ethanol (dielectric constant: 2
4.30), n-butanol (dielectric constant: 17.1), and other alcohol solvents, acetone (dielectric constant: 20.7)
4) etc. Ketone solvents, diethyl ether (dielectric constant:
4.335) and other ether solvents, ethyl acetate (dielectric constant: 6.02) and other ester solvents, acetic acid (dielectric constant:
6.15) and other organic acid-based solvents and derivatives thereof, hydrocarbon solvents such as cyclohexane (dielectric constant: 2.023),
Benzene (dielectric constant: 2.284), toluene (dielectric constant:
2.38) and other benzene ring solvents, pyridine (dielectric constant:
12.3) and other organic salt solvents, and acetonitrile ((dielectric constant: 37.5) and other cyanide solvents, and water (dielectric constant: 80.1). These polar solvents include The polar solvent used in the present invention is distilled off after the extraction operation, so that a solvent having a low boiling point is used as long as it has the same solubility property. It is preferable to use an organic polar solvent having a boiling point of usually 80 ° C. or lower, preferably 60 ° C. or lower as the organic polar solvent among the above polar solvents.

In the present invention, the extraction process using a polar solvent as described above may be carried out in multiple stages using polarities having different dissolution characteristics. For example, in FIG. 1, the deep water-soluble component separated from the deep sea water was extracted with chloroform, the residue residue was extracted with methanol, and the methanol extraction residue residue was extracted with 80% ethanol. An example of extracting the 80% ethanol extraction residue with hot water is shown.

10 to 100 ml, preferably 20 to 10 g of deep water-soluble components separated from the deep sea water
An amount of ˜50 ml of polar solvent is used in one extraction, and the extraction operation with one kind of extraction solvent is usually performed 1 to 6 times, preferably 1 to 5 times. Then, for example, by sequentially extracting from a solvent having a high polarity using a solvent having a low polarity, it is also possible to fractionate and extract a deep-water-soluble component having a polarity corresponding to the polarity of the solvent used. . If the amount of extraction solvent used is too large, it will take a long time to remove the solvent after extraction, and the amount of calcium sulfate, which is the main component of the deep-water soluble components separated from the deep sea water, to the extract The amount of migration increases.

In this extraction, the deep water-soluble component separated from the deep sea water containing the component to be extracted is brought into contact with the extraction solvent, and the mixture of both is subjected to ultrasonic stirring, mechanical stirring, etc. The component to be extracted is transferred to a polar solvent. The contact conditions between the two can be appropriately set depending on the solvent and the component to be extracted, but the temperature is not higher than the boiling point of the solvent to be used, and usually contacted for 1 minute to 24 hours, preferably for 10 minutes to 10 hours, and then By filtering this mixture, the soluble component in the solvent used is dissolved in the filtrate and separated from the insoluble component in the extraction solvent. For this separation operation, individual liquid separation methods such as centrifugation, filtration and decantation can be adopted. For example, when separating the extraction solvent and the solid content after the solvent extraction by a filtration operation, a membrane filter having an opening of about 0.01 to 2 μm can be used.

For example, a substance having eosinophil activity is separated from the deep water soluble component separated from the deep sea water by removing the extraction solvent from the filtrate separated using the membrane filter or the like as described above. can do. When removing the extraction solvent, it is preferable to remove the extraction solvent at a temperature as low as possible so that the extract is not thermally decomposed. Since the extraction solvent as exemplified above has a relatively low boiling point, it is preferable to remove the extract by a method that does not heat the extract as much as possible, such as removal under reduced pressure.

The residue after the extraction treatment with the polar solvent as described above was sufficiently dried, and the infrared absorption spectrum (2) measured on the residue was shown in FIG.
Shown in. The infrared absorption spectrum (2) chart shown in FIG. 4 has a small peak near 1438 cm ^-1 , but is very similar to the infrared absorption spectrum (3) chart of the calcium sulfate special grade reagent shown in FIG. It is an approximate pattern, and it is considered that most of the substance represented by the chart of the infrared absorption spectrum (2) shown in FIG. 4 is calcium sulfate. Furthermore, the infrared absorption spectrum (2) shown in FIG. 4 measured on the residue obtained by performing the extraction operation as described above, and the deep layer separated from the deep sea water before the extraction operation shown in FIG. When comparing the infrared absorption spectrum (1) measured for the water-soluble components, 871cm ^-1 was present in the infrared absorption spectrum ^{(1), 1406cm -1, 1477cm} -1, 1
It can be seen that the peaks near 540 cm ^-1 , 2113 cm ^-1 and 2974 cm ^-1 have almost disappeared in the infrared absorption spectrum (2). Therefore, it is considered that the substances having these peaks were transferred to the polar solvent phase and separated by the extraction operation, and most of the residue from the extraction operation using the polar solvent was calcium sulfate. it is conceivable that.

Therefore, the results of examining the eosinophil activity of the components separated by the extraction operation using the polar solvent are shown in FIG. Although the eosinophil activity of the extracted components varies depending on the polar solvent used, when the extraction operation is performed in this order using, for example, chloroform, methanol, and ethanol, the chloroform extract becomes a control (PBS: phosphate buffer physiological). Saline solution) shows about 4.6 times more eosinophil activity, and the methanol extract shows about 2.2 times more eosinophil activity than the control (PBS) and ethanol extract. The product shows about 5.6 times more eosinophil activity than the control (PBS).

A hot water extract extracted with hot water (usually 80 to 100 ° C. or less) after extraction using the above organic polar solvent, and 10 wt% NaOH of this hot water extraction residue The NaOH extraction part, which was obtained by neutralizing the extract with hydrochloric acid and dialyzing it, is about 2 times or less the control (PBS),
Shows eosinophil activity. In addition, the positive control (O
Z: opsonized zymosan (immunostimulator), the chloroform extract showed about 2.3 times the eosinophil activity, and the methanol extract showed about 1.2 times the eosinophil activity. The ethanol extract shows about 2.7 times the eosinophil activity.

The component exhibiting such eosinophil activity is originally a component dissolved in deep sea water, and the component deposited by concentrating the deep sea water is brought into contact with a polar solvent to polar solvent. It is a substance eluted by, and contains a wide variety of components, and it is extremely difficult to specify these. However, since it is originally dissolved in deep sea water and is a substance that can be extracted by a polar solvent, particularly an organic polar solvent, it is highly likely that it is a mixture of organic compounds having polar groups. Alternatively, deep sea water does not undergo photosynthesis naturally because it flows through the very deep sea floor where sunlight does not reach, but products, secretions, decomposition of aquatic plants, aquatic animals, etc. that grow in ocean water. It is fully conceivable that organic substances such as substances are contained and accumulated in the deep sea water, and it is also possible that such organic substances and complexes of organic substances and inorganic substances are dissolved in the deep sea water.

Although the details of the compound name, structure, composition, etc. of these extracts showing eosinophil activity have not been clarified, their molecular weight is not so large because they are substances extracted and separated by the above-mentioned separation operation. It is presumed to be an organic compound. Then, it is considered that these organic compounds act in a complex manner, and abundantly dissolved minerals and the like interact to exert excellent eosinophil activity as shown in FIG.

As described above, in an allergic reaction such as atopic dermatitis, locally-migrated eosinophils release histamylase, allylsulfatase B and phospholipase D ₂ . These released substances are eosinofhil chemotactic factor of anaphylaxis, which is an abnormal hypersensitivity reaction to foreign substances such as histamine and heterologous proteins that mediate inflammation.
And platelet activating factor
(PAF)) and is known to work to repair allergic reactions. Furthermore, recently, eosinophils have come to be considered to act on tissue damage more than the above-mentioned repair action of the tissue. Four basic proteins (major basic pr) present in special granules of eosinophils play a role in the involvement of eosinophils in tissue damage.
otein (MBP), eosinophil peroxidase (MPO), eosinopil
combined protein (ECP), eosinopil derived neurotoxi
n (EDN)) and superoxide (O ₂ ^- ) ^- derived various active enzyme molecular species produced by eosinophils. These four
It is believed that a class of basic proteins are released from activated eosinophils by degranulation into tissues and are involved in tissue damage. In particular, it is considered that MBP activates basophils and MPO activates mast cells to release histamine to enhance allergic reaction.

Further, superoxide (O ₂ ^-) is, eosinophils IgE complex, PAF, opsonized zymosan (O
Z), phorbol myristate acetate, and various other stimuli produce NADPH oxidase, which is a cell membrane-bound enzyme system that is activated. This NADPH oxidase oxidizes NADPH produced by a sharp increase in hexose monophosphate shunt activity, thereby producing O ₂ ⁻ in which oxygen is reduced by one electron. From this O ₂ ^−, various reactive oxygen molecular species having high reactivity such as H ₂ O ₂ , HO ⁻ and ¹ O ₂ are generated by an enzymatic or non-enzymatic reaction. These reactive oxygen species are responsible for important biological defense such as sterilization. Therefore, the ability of eosinophils to promote the production of superoxide (O ₂ ⁻ ) is very useful for suppressing the causative factors of allergic reactions such as atopic dermatitis.

[0036] Then, superoxide generated by the marine from components dissolved in deep water using the extracted separated components with a polar solvent as described above eosinophils ^- peroxide oxidized (O ₂₎ When the hydrogen production capacity was measured, as shown in FIG. 5, the substances extracted and separated using any polar solvent were able to activate eosinophils more efficiently than the control (PBS), and the chloroform extract, The methanol extract and 80% ethanol extract can activate eosinophils more efficiently than the positive control (OZ). In particular, the degree of activation of eosinophils of the chloroform extract and the 80% ethanol extract is extremely high.

In the above description, since the extraction solvent is chloroform, methanol, and 80% ethanol in this order, the activity of the chloroform extract is higher than that of the methanol extract. From the relative relationship of the dielectric constant values, it is estimated that most of the chloroform extract is extracted with methanol if methanol is directly extracted without chloroform extraction. The degree of sphere activation is estimated to be higher than the degree of eosinophil activation of the methanol extract shown in FIG.

In the present invention, the degree of activation of eosinophils can be measured, for example, by the method described below. That is, for example, a growth differentiation factor that is selective for eosinophils
Transgenic mice that constantly produce IL-5 (C3
H / HeN-TgN (IL-5) Imeg) 15-20 weeks old spleen eosinophils and spinal cord eosinophils were prepared.
²⁺ Free Krebs-Ringer-phospate buffer solution (KR
P) (pH value 7.4) can be homogenized and the cell suspension can be separated by a method such as Percoll discontinuous density gradient centrifugation.
Usually, 75% of the spleen and 80% of the spinal cord can be used as the eosinophil fraction. The mouse used here is preferably one in which 20 μg of cadmium sulfate was intraperitoneally administered 5 days before the experiment for the purpose of increasing IL-5 production under the control of the metallothionein promoter.

The ability of the eosinophil active ingredient of the present invention contained in deep sea water to enhance the production of superoxide (O ₂ ⁻ ) of eosinophils is reduced by hydrogen peroxide produced by eosinophils. It can be calculated by measuring the amount of cytochrome c (Cyt. C). This measurement is from Nakagaw
A. A., Shibata. K., Takeshige. K., et al: Exp.Cel
l.Res. 101: 224-234 (1976).

That is, for example, 20 μmol of cytochrome
Mu c (Cyt. C), 10 mmol glucose, 1 mM CaC
l₂The eosinophils (5
× 10 ^Fivecells / ml), and 550 accompanying the reduction of Cyt.c
Increase in absorbance at nm (A_550-540) Is measured over time. Na
Oh, O₂ ^-The amount of produced is Cyt. C molecular absorbance 21.0 mM.
^-1·cm^-1Can be obtained from Also, for this measurement
Is a device that can measure absorbance while stirring the reaction solution (eg
For example, a two-wavelength spectrophotometer (UV-30 manufactured by Shimadzu Corporation)
0)) is preferably used.

The degree of activation of eosinophils can be measured by the method described below. That is, for example, it can be calculated by measuring the amount of hydrogen peroxide (H ₂ O ₂ ) derived from superoxide (O ₂ ⁻ ) produced by eosinophils. The amount of hydrogen peroxide (H ₂ O ₂ ) indicates the hydrogen peroxide-producing ability of eosinophils, and can represent the degree of activation of eosinophils as an activation index. This measurement is
Irene, AMVint, John, C. Foreman, et al: Eur. J. I
mmunol. 24: 1961-1965, 1994.

[0042] That is, superoxide eosinophils produced activated by the active ingredient of deep seawater (O ₂ ^-)
The amount of hydrogen peroxide (H ₂ O ₂ ) derived from
Fluorescence from 7'-dichlorodihydrofluorescein diacetate (DCFH-DA) can be analyzed using a flow cytometer. Specifically, 5 × 10 ⁵ cells / ml of eosinophils were added to Phosphhate-buffer saline containing 2.5 μmol DCFH-DA.
37 in 0.5 ml glucose (5 mM glucose) (PBSg)
Incubate at 15 ° C for 15 minutes, then add stimulant and incubate at 37 ° C for 30 minutes, and finally PB
After washing the cells several times with Sg, it can be analyzed and measured with a flow cytometer.

The eosinophils used above are, for example, transgenic mice (C3H / HeN-TgN (IL-5) Imeg) that constantly produce IL-5, which is a growth differentiation factor selective for eosinophils. )
Eosinophils isolated from the peripheral blood of can be used in the experiment. The above-mentioned measurement method is an example of a method for measuring the activation degree of eosinophils, and the activation degree of the eosinophil-active substance of the present invention is not limited to the above method.

The drug containing the eosinophil active substance of the present invention is highly useful as a component of a drug used for allergic diseases, especially atopic dermatitis. That is, the eosinophil activator of the present invention activates locally-migrated eosinophils to cause abnormal hypersensitivity reactions to foreign substances such as histamine and heterologous proteins in allergic reactions, eosinofhil chemotactic factor. of anaphylaxis)
And platelet activating facto
It releases histamylase, allylsulfatase B, and phospolypase D ₂ which metabolize r (PAF). Furthermore, the bioprotective reaction can be improved by the formation of superoxide (O ₂ ⁻ ) by the activation of the cell membrane-bound enzyme system by eosinophils.

The eosinophil active substance of the present invention can also be used as an external preparation dissolved or dispersed in a base material.
When used as an external preparation, the amount of the eosinophil active substance in the external preparation is usually 0.00001 to 1
It is 0.0% by weight, preferably 0.001 to 0.1% by weight. In this case, as a base material for the external medicine, petroleum jelly, ceresin, paraffin, squalane, waxes, ozokelide, and the like which are used as the base material for the usual external medicine can be used. Further, as a dosage form, it can be applied to an emulsion form, an oily form, a wax form, a lotion form and the like.

Further, the eosinophil-active substance of the present invention can be used as an injection by making it into an aqueous solution. The concentration of the eosinophil-active substance in this case is usually 0.0
00001 to 10.0% by weight, preferably 0.0001
It is set within the range of 0.1% by weight. When used as an injectable solution, it is possible to add components such as a pH adjusting agent and an antioxidant, which are added to ensure stability of the injectable solution.

Furthermore, the eosinophil-active substance of the present invention can be used as an internal medicine such as powders, tablets, granules, capsules and syrups. When used as an internal medicine using the eosinophil active substance of the present invention, an injection, the amount of use can be appropriately set in consideration of symptoms, the condition of the user, etc., but the amount used per 1 kg of body weight is: In general,
0.0001-10000 mg, preferably 0.1-10
It can be set within the range of 0 mg.

[0048]

Industrial Applicability The eosinophil active substance of the present invention is a component dissolved in deep ocean water, and is a substance which can be extracted from polar substances after being precipitated from deep ocean water. This substance can be extracted with a polar solvent and is considered to be an organic compound having a not so large molecular weight or a complex containing such an organic compound as a main component. This substance is
It can activate eosinophils and has a very excellent biological defense function by the action of superoxide produced by the activation of eosinophils. This substance also metabolizes histamine, which mediates inflammation, and releases histamylase, which works to repair allergic reactions.

Further, the eosinophil-active substance is an extraction solvent which is once precipitated by concentrating and desalting the components dissolved in the deep sea water, and the precipitate is extracted with a polar solvent. a component obtained by removing the polar solvent, these extraction components, superoxide from eosinophils (O ₂ ^-) is generated, the superoxide ^- enzymatic reactions or nonenzymatic from (O ₂₎ Various reactive oxygen species having high reactivity can be generated by the reaction. Then, these active oxygen species can express important biological defense such as bactericidal action.

The above-mentioned functions of the eosinophil active substance of the present invention act in combination, so that the eosinophil active substance of the present invention can be used for allergic diseases such as asthma, hay fever, and dermatitis. It is highly useful as a therapeutic component, especially as a therapeutic component for allergic diseases such as atopic dermatitis. Further, the eosinophil active substance of the present invention can be obtained by precipitating a component dissolved in deep sea water and then extracting with a polar solvent, and these operations are separation operations. Since the reaction of the compound essentially does not proceed during the operation of, the component contained in the deep sea water from the beginning and capable of improving the activity of eosinophils can be selectively obtained. Moreover, since the boiling point of the extraction solvent used is not so high, the extraction solvent (polar solvent) can be easily removed by reducing the pressure, for example.

Furthermore, since the eosinophil active substance of the present invention has a good affinity for water and also an affinity for organic compounds so that it can be extracted with an organic polar solvent, it may be used as an external drug, an internal drug, an injection solution, etc. It can be used in various forms. Moreover, such an eosinophil active substance of the present invention has remarkably few side effects as compared with a normal eosinophil active substance, and is extremely high in safety, especially a therapeutic agent for allergic diseases such as atopic dermatitis. As very useful.

[0052]

The present invention will be described in more detail with reference to the following examples, which should not be construed as limiting the invention.

[0053]

[Example 1] [Preparation of eosinophil active substance] 20 liters of deep sea water collected from a deep sea at a depth of 400 m was opened.
The suspension was removed by filtration using a 45 μm membrane filter. The deep sea water filtered in this way is put into a vacuum distillation apparatus, and the distillation temperature is set to a temperature of 40 ° C. or lower under a pressure condition of a pressure reduction degree of 20 Torr, and the amount of deep sea water is about 2.
It was distilled under reduced pressure to 1 liter (1/10 of the original ocean water).

Since the components dissolved in the deep sea water were precipitated by the vacuum distillation in this way, the precipitate (1) was collected by filtration with a membrane filter having an opening of 0.45 μm. On the other hand, the filtrate obtained by filtering as described above is a mosaic charged membrane (trade name: MC-55B
Using a mosaic charged film, manufactured by Tosoh Corporation, low temperature 10 ° C
The desalting operation was carried out under the conditions described above, and the desalted solution was placed in a vacuum distillation apparatus, and the temperature was controlled so that the temperature did not exceed 40 ° C. under a reduced pressure of 20 Torr.
Water was distilled off under reduced pressure until the volume became ml.

Desalting using the above-mentioned mosaic charged membrane and distillation under reduced pressure were repeated to concentrate to 5 ml. The precipitate (2) thus deposited by repeating desalting and distillation under reduced pressure was collected by filtration. The total amount of the precipitates (1) and (2) collected by filtration as described above was 19.18 g. The infrared absorption spectrum of the mixture of the precipitates (1) and (2) was measured. The results are shown in Figure 2.

5 g of the above-mentioned precipitate was collected, and this precipitate 5
g was put in 50 ml of chloroform (dielectric constant: 4.86), and the temperature of this dispersion was maintained at 50 ° C. or lower and ultrasonically stirred for 30 minutes. Then, the dispersion liquid was filtered using a membrane filter having an opening of 0.45 μm to separate the filtrate from the solid matter. The same operation as above is performed twice more on the solid matter separated as described above.
0 ml of chloroform filtrate was obtained. The chloroform filtrate was heated to 40 ° C. under a reduced pressure of 20 Torr to remove chloroform to obtain 25.2 mg of chloroform extract.

Then, the solid matter separated by the membrane filter by the above-mentioned chloroform extraction was extracted with methanol. That is, in the above chloroform extraction, methanol extraction was carried out in the same manner except that the extraction solvent was changed to methanol (dielectric constant: 32.65).
To obtain a methanol filtrate of. Methanol was removed from this methanol filtrate in the same manner as in the case of chloroform to obtain 9.3 mg of a methanol extract.

Further, 80% ethanol (ethanol: 80% by volume, water: 20% by volume, dielectric constant value: 35.46) was extracted using the solid substance separated by the membrane filter by the methanol extraction. That is, in the above extraction with chloroform, 80% ethanol extraction was performed in the same manner except that the extraction solvent was changed to 80% ethanol to obtain 150 ml of 80% ethanol filtrate.

Ethanol and water were removed from this 80% ethanol filtrate in the same manner as in the case of the above chloroform to obtain 1
0.7 mg of 80% ethanol extract was obtained. On the other hand, after performing 80% ethanol extraction as described above, the infrared absorption spectrum (2) was measured for the solid content on the membrane filter. The results are shown in Fig. 4. Separately from this, the infrared absorption spectrum of a commercially available calcium sulfate special grade reagent (manufactured by Wako Pure Chemical Industries, Ltd.) is shown in FIG.

Comparing FIG. 2 and FIG. 4, the infrared absorption spectra of FIG. 2 show 871 cm ⁻¹ , 1406 cm ⁻¹ , 14
^{77cm -1, 1540cm -1, 2113cm -1} , 2974cm -1
The difference is that there are peaks in the vicinity. The infrared absorption spectrum shown in FIG. 4 shows the precipitates (1) and (2) above.
The mixture with and was extracted with chloroform, methanol and 80% ethanol as described above. The infrared absorption spectrum shown in FIG. 4 and the infrared absorption spectrum shown in FIG. 3 have peaks that are very similar. The infrared absorption spectrum of FIG. 3 has a purity of 99.
It is a spectrum of highly pure calcium sulfate of 9% or more, and most of the compounds represented by the infrared absorption spectrum of FIG. 4 are considered to be calcium sulfate. Therefore, the infrared absorption spectrum shown in FIG.
The difference between the infrared absorption spectrum shown in and
The components contained in the mixture of the precipitates (1) and (2) showing the infrared absorption spectrum shown in FIG. 2, that is, those derived from the extract obtained by the above chloroform, methanol and 80% ethanol extraction. Is estimated.

After 80% ethanol extraction was carried out as described above, the solid content on the membrane filter was put into 50 ml of hot water at 95 ° C. and ultrasonically stirred for 30 minutes.
It was filtered using a membrane filter having an opening of 0.45 μm. The above procedure was repeated twice more to obtain 150 ml of filtrate. Water was removed from this filtrate by distillation under reduced pressure, and
2 mg of hot water extract was obtained.

On the other hand, after the above hot water extraction, the solid content on the membrane filter was changed to 10% sodium hydroxide aqueous solution 5
After pouring into 0 ml and stirring at room temperature for an hour, the opening is 0.45
It was filtered using a μm membrane filter. 4.51 g of solid content filtered by membrane filter
Met. On the other hand, the 10% aqueous sodium hydroxide solution extract (filtrate) was neutralized with hydrochloric acid and dialyzed to obtain 8.5 mg of a NaOH extract. [Activation degree of eosinophil active substance extracted from deep sea water] The eosinophil activation degree of the eosinophil active substance of the present invention contained in deep sea water is eosinophil Was measured using the amount of hydrogen peroxide (H ₂ O ₂ ) derived from superoxide (O ₂ ⁻ ) produced by the above as an index. Specifically, eosinophils superoxide produced activated by the active ingredient of deep seawater (O ₂ ^-) Measurement of the amount of hydrogen peroxide derived from (H ₂ O ₂₎ is, 2 ', 7 '-dichlorodihydrofluor
Fluorescence by escein diacetate (DCFH-DA) was analyzed using a flow cytometer. Phosphhate-buffer sa containing 2.5 μmol DCFH-DA containing 5 × 10 ⁵ cells / ml of eosinophils
3 in 0.5 ml of line glucose (5 mM glucose) (PBSg)
Incubate at 7 ° C for 15 minutes, then stimulate (P
MA (50 ng / ml) was added, and the mixture was incubated at 37 ° C for 30 minutes. After washing the cells twice with PBSg, the cells were analyzed and measured with a flow cytometer.

The eosinophils used in the above were the transgenic mice (C3H / HeN-TgN (IL-5) Imeg) that constitutively produce IL-5, which is a growth differentiation factor selective for eosinophils. Eosinophils isolated from peripheral blood were used in the experiment. Peripheral blood Ca ⁺⁺ free containing 0.22% final concentration, Krebs Ring
er phosphate buffer (KRP) (0.9% NaCl, 6 mM KCl, 1 mM MgCl ₂ , 10 mM Na-phosphate buffer (pH 7.4) for the purpose of removing red blood cells and monocytes Percoll discontinuous density-gradient centrifugation was performed at 10. The concentration of KRP was 10 times that of the stock solution of Percoll.
10% added, 100% Percoll definition 60, 70, 80% Percoll overlaid under cell turbidity, 350g,
After centrifugation at room temperature for 20 minutes, 70 and 80% layers were collected. The collected cell population was treated with a separation buffer (5 mmol EDT).
A, 0.5% BSA, 10 mmol mol Na-K-phosphate buffer (pH 7.2)) and further suspended (1 x 10 ⁷ cells /
50 μl), and anti-Thy1.2 antibody-conjugated magnetic beads (1
× 10 ⁷ beads / 25 μl) and anti-B220 antibody-bound magnetic beads (1 × 10 ⁷ beads / 25 μl), and added at 4 ° C. for 2
After reacting for 0 minutes, a cell population collected by negative selection using a permanent magnet was subjected to an experiment as an eosinophil population. The purity of the cell population after separation was measured by a flow cytometer (FECT manufactured by BECTON DICKINSON).
It was analyzed by ACSscan).

As the substance to be measured, the liquid component is sample / 1.8
% NaCl and purified water, which was adjusted to be isotonic was added at a final concentration of 100 μg / ml. The solid component was suspended in physiological saline and added at a final concentration of 100 μg / ml. The eosinophil activation value (corresponding to the hydrogen peroxide production amount) of each test substance measured as described above is shown in FIG. Also, as an example,
FIG. 6 shows the results of flow cytometer analysis and measurement of the hydrogen peroxide production amount of eosinophils of the control (PBS) and the hydrogen peroxide production amount of eosinophils of the chloroform extract.

As shown in FIG. 5, the control (PB
S) has a degree of eosinophil activation of 3.35, while the chloroform extract has about 4.6 times the degree of activation of eosinophils (15.
68) eosinophil activity is shown, and the methanol extract is
The eosinophil activity was about 2.2 times that of the control (PBS) (the eosinophil activation degree was 7.6), and the 80% ethanol extract was about 5 times the control (PBS). ．
The eosinophil activity was about 6-fold (eosinophil activation degree 18.62).

After extraction with an organic polar solvent,
A hot water extract extracted with hot water, and a 10 wt% NaOH extract of this hot water extraction residue, which was neutralized with hydrochloric acid and dialyzed
The extraction portion also showed eosinophil activity, although it was about twice as much (eosinophil activation degree 5.88 and 6.35) as compared to the control (PBS). In addition, positive control (OZ:
The eosinophil activity of opsonized zymosan (eosinophil activation degree 6.79) was about 2.
The eosinophil activity was about 3-fold, the methanol extract showed about 1.2-fold eosinophil activity, and the ethanol extract showed about 2.7-fold eosinophil activity. From the above results, it was revealed that the polar solvent-soluble matter contained in the deep sea water obtained in the present invention has eosinophil activity for eosinophils.

[Brief description of drawings]

FIG. 1 is a process drawing showing an example of a method for obtaining an eosinophil active substance of the present invention.

FIG. 2 is a chart of an infrared absorption spectrum (1) of a deep water soluble component separated from deep sea water.

FIG. 3 is a chart of infrared absorption spectrum (3) of a special grade reagent of calcium sulfate.

FIG. 4 is a chart of infrared absorption spectrum (2) of this residue after extraction treatment with a polar solvent and drying.

FIG. 5 is a graph showing the degree of activation of the eosinophil-active substance of the present invention.

FIG. 6 is a chart of hydrogen peroxide producing ability of eosinophils analyzed and measured by a flow cytometer.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61P 43/00 111 A61P 43/00 111

Claims (11)

[Claims] 1. An eosinophil active substance contained in a polar solvent-soluble component which is transferred from the deep-water-soluble substance to the polar solvent phase by contact between the deep-water-soluble substance separated from the deep-sea water and the polar solvent. 2. The dielectric constant of the polar solvent is 2.0 to 200.
The eosinophil active substance according to claim 1, wherein the eosinophil active substance is in the range. 3. The eosinophil active substance according to claim 1, wherein the deep sea water is collected from a deep sea having a depth of 200 m or more. 4. A precipitate from the concentrated deep sea water, which is obtained by concentrating the deep sea water separated from the deep sea water to 1/5 to 1/1000 of the initial amount of deep sea water and further desalting. The eosinophil-active substance according to claim 1, which is 5. The eosinophil active substance does not transfer to the polar solvent phase obtained after contact with the infrared absorption spectrum (1) measured for deep-layer water-soluble substances separated from deep-sea water and the polar solvent. By comparison with the infrared absorption spectrum (2) measured for the component, a substance characterized by an infrared absorption spectrum peak that is present in the infrared absorption spectrum (1) but not in the infrared absorption spectrum (2) The eosinophil active substance according to claim 1, which is characterized by being present. 6. A step of separating a substance dissolved in deep-sea water from the deep-sea water, a step of bringing the separated deep-water soluble matter into contact with a polar solvent, and the polar contained in the deep-water soluble matter. A step of transferring a solvent-soluble substance to a polar solvent phase, a step of separating the polar solvent phase and a component insoluble in the polar solvent, and a polar solvent soluble by removing the polar solvent from the separated polar solvent phase. A method for obtaining a high concentration of an eosinophil active substance, comprising the step of obtaining an eosinophil active substance contained in a component. 7. The method for obtaining an eosinophil active substance according to claim 6, wherein the polar solvent has a dielectric constant in the range of 2.0 to 200. 8. The method for obtaining a high-concentration eosinophil-active substance according to claim 6, wherein the deep sea water is collected from a deep sea having a depth of 200 m or more. 9. Deep sea water in a volume ratio of 1/5 to 1/10
7. The eosinophil-active substance according to claim 6, wherein the substance dissolved in the deep sea water is separated through a step of desalting the concentrated deep sea water. How to get by concentration. 10. The separated deep-layer aqueous solution is sequentially brought into contact with a plurality of polar solvents having different dielectric constants, and polar solvent-soluble components contained in the deep-layer aqueous solution are sequentially transferred to the polar solvent layer. The method for obtaining the eosinophil-active substance according to claim 6 at a high concentration. 11. An eosinophil-active substance contained in a polar solvent-soluble component which migrates from the deep aqueous solution to the polar solvent phase by contact between the deep aqueous solution separated from deep sea water and the polar solvent. Pharmaceuticals characterized by: