JP2010270104A - iNKT CELL ACTIVATOR - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To identify a novel ligand of iNKT cells concerning a purified fraction of sphingoglycolipid obtained from a plant material such as rice bran and rice germ, to elucidate its mechanism of action, to investigate its effectiveness against various pathological conditions (infections, tumors, allergies, etc.), and to thereby provide the possibility of application of it to drugs and diagnostic drugs, namely, to provide iNKT cell activators using the purified fraction. <P>SOLUTION: There are provided iNKT cell activators comprising rice bran, rice germ and/or sphingoglycolipids derived therefrom; the iNKT cell activators comprising a sphingoglycolipid purified fraction prepared by fractionating a sphingoglycolipid-containing crude fraction obtained by extracting rice bran, rice germ, and/or a material derived therefrom with an organic solvent by chromatography and/or recrystallization; and functional foods, functional health drinks, food additives, cosmetics, quasi-drugs, drugs, diagnostic drugs, reagents for biochemical studies, etc., each of which is based on the iNKT cell activation action. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to an iNKT cell (invariant natural killer T cell) activator comprising a sphingoglycolipid obtained from a plant material such as rice bran, rice germ, and the like, and more specifically, comprising such an iNKT cell activator. The present invention relates to pharmaceuticals, particularly immune enhancers, anticancer agents, antiallergic agents, infection resistance enhancers, and further novel iNKT cell ligands.

  Rice has long been the most widely grown cereal resource in Japan as a Japanese staple food. Rice is rarely edible as brown rice, and is used as edible rice after milling. So far, a large amount of rice bran produced in the rice milling process has been partly used as feed and fertilizer, but most of it has been treated as agricultural waste.

  In recent years, lecithin, ceramide, etc. from gums, which are produced in the process of refining rice oil from rice crude oil (derived from rice bran), higher fatty acids, etc. from wax, γ-oryzanol, ferulic acid, etc. from dark oil, scum (scum) : Tocotrienol, Vitamin E and many other useful physiologically active substances have been isolated and refined from Floating Mochi) and have been widely used as pharmaceuticals, quasi drugs, cosmetics, food additives, foods, etc. .

  The defatted koji produced after squeezing rice oil contains physiologically active substances such as GABA, phytic acid, and inositol, and is used as a useful natural physiologically active substance. In particular, rice ceramide (comedosphingoglycolipid) is composed of various β-glucosylceramides (β-GluCer), and has a moisturizing effect and blood lipid lowering action like animal ceramides. Recently, it has been suggested that it may be involved in lipid metabolism (Patent Document 1).

  Furthermore, not only comeseramide (comesphingoglycolipid) but also NKT cell activation composition, IL-4 production promotion composition, IFN-γ production promotion composition, dendritic cell activation containing glycosphingolipid. Composition, IL-12 production promoting composition, IL-10 production promoting composition, NK cell activation composition, antitumor composition, antiallergic composition, infection resistance enhancing composition, Proposals have also been made regarding antiviral composition, IL-6 production promoting composition, NO production promoting composition, etc. (Patent Document 2).

Furthermore, a method for producing a glycosphingolipid-containing product using plant materials as a raw material (Patent Document 3) and a preventive agent for colorectal cancer containing a glycosphingolipid have also been proposed (Patent Document 4).
On the other hand, glycosphingolipids derived from non-food sponges (α-galactosylceramide; α-GalCer) are known to have an iNKT cell activation effect, and have been pointed out as potential immunopotentiators and anticancer agents. (Patent Document 5).

However, there has been little elucidation of iNKT cell activation action of rice ceramide, especially glycomesphingoglycolipid, or ligand of iNKT cell, and any component of comemesphingoglycolipid which is a mixture is active. It is not clear at all about whether to show.
International Patent Publication No. WO2005 / 068041 Japanese Patent Laying-Open No. 2005-267397 JP 2005-120321 A JP 2005-187391 A JP 2006-290856 A Science, 278: 1626, 1997 Annu. Rev. Immunol., 23: 877, 2005 Proc. Natl. Acad. Sci. USA, 102: 1351, 2005 Nature, 434: 520, 2005 Nature, 434: 525, 2005 Eur. J. Immunol., 35: 1692, 2005 Nat. Immunol., 7: 978, 2006 Science, 306: 1786, 2004 Nature, 434: 525, 2005 Proc. Natl. Acad. Sci. USA, 104: 5977, 2007

At present, there are few preventive measures against cancer (tumor) and it is left to treatment only. The treatment method is performed by combining various anticancer (tumor) agents, radiation, surgical removal, or any combination, but there are strong side effects in the case of anticancer agents, and in the case of radiation therapy That there is no effective treatment method because of the cost of surgery, the considerable burden on patients when surgically removed, and the possibility of recurrence even after these treatments is the current situation.
In this way, in addition to the limitations of existing preventive and therapeutic methods for cancer (tumors), stress in dietary and daily life is increasing rapidly, and the number of cancer diseases is increasing worldwide. Therefore, there is an urgent need to develop a new preventive / therapeutic that can be safely, reliably and simply provided for cancer (tumor).

  Until now, vaccines have been widely used as a preventive measure against various infectious diseases. Vaccines are based on the fact that the specific immune response against the target pathogenic microorganism lasts for a long time, but the duration of the effect varies greatly depending on the target pathogenic microorganism, depending on the pathogen, In addition, since individual differences may occur, it is not necessarily an effective preventive measure. In addition, the live vaccine may mutate the attenuated strain used, which is problematic in terms of safety.

In general, antibiotics such as antibacterial agents, antifungal agents, and antiviral agents are used as therapeutic agents in the case of suffering from infectious diseases. Antibiotics are administered to reduce (or kill) microorganisms in the body without destroying human cells, and to kill and eliminate pathogenic microorganisms remaining by the immune response of the living body.
However, in recent years, multidrug-resistant bacteria have emerged due to heavy use of antibiotics, which has become a social problem such as nosocomial infections and opportunistic infections, so urgent measures are required.
In this way, in addition to the limitations of existing preventive and therapeutic methods for infectious diseases, the number of affected people with re-emerging infectious diseases and emerging infectious diseases is increasing worldwide, so it can be provided safely, reliably and easily. There is an urgent need to develop new preventive and therapeutic drugs.

  Under such circumstances, the present inventors have used iNKT cells for a purified fraction containing a high level of glycosphingolipid obtained from rice bran for the purpose of effective utilization of rice bran that has been treated as agricultural waste. Functional ligands, functional health drinks, food additives, cosmetics, quasi-drugs, pharmaceuticals, diagnostics, biochemical research by isolating and identifying new ligands and clarifying their mechanism of action A study was conducted to clarify the usefulness of the reagent as a pharmaceutical reagent.

  In other words, the present invention isolates and identifies a novel ligand of iNKT cells from a purified fraction of glycosphingolipid obtained from plant materials such as rice bran and rice germ, and elucidates the mechanism of its action, To examine the effectiveness of the product for infectious diseases, tumors, allergies, etc.) and provide the possibility of application to pharmaceuticals and diagnostics, that is, to provide an iNKT cell activator using this purified fraction Let it be an issue.

  As a result of intensive investigations by the present inventors in order to solve such problems, the present inventors have obtained a glycosphingolipid-containing purified fraction obtained by separating and purifying an organic solvent extract fraction of plant material such as rice bran and rice germ. It has been confirmed that there is a strong iNKT cell activation action, and therefore there is an immune enhancement action through such iNKT cell activation action, particularly a cancer metastasis suppression action, an infection resistance enhancement action, etc. It came to complete.

  Thus, the present invention for solving the above-described problems is an iNKT cell activator comprising rice bran, rice germ and / or glycosphingolipid derived therefrom as a basic aspect thereof.

  Specifically, the present invention relates to a glycosphingolipid-containing crude fraction obtained by extracting a glycosphingolipid with rice bran, rice germ, and / or a material derived therefrom using an organic solvent, by chromatography and / or recrystallization. The iNKT cell activator described above, characterized by comprising a fractionated glycosphingolipid purified fraction.

  More specifically, the iNKT cell activator described above, wherein the organic solvent extraction is alcohol, ketone, chlorinated hydrocarbon and / or mixed solvent thereof, and / or water and mixed solvent extraction thereof.

  More specifically, the iNKT cell activator described above, wherein the chromatography is column chromatography or liquid chromatography.

  Moreover, this invention is said iNKT cell activator whose recrystallization is alcohol, a ketone, and these mixed solvents, and / or water and these mixed solvents.

  For example, in the present invention, more specifically, the organic solvent extraction is ethanol extraction, the chromatography is silica gel column chromatography, and the elution solvent for column chromatography is successively chloroform / chloroform-acetone mixed solution / methanol. It is an iNKT cell activator comprising a purified glycosphingolipid fraction obtained by elution.

  Among the fractions thus purified, glycosphingolipid is present in the methanol-eluting portion and can be used as it is, but further purification steps can be added.

  That is, a glycosphingolipid, which is a white crystalline powder obtained by recrystallizing a methanol-eluted fraction with methanol, is more highly active.

  Alternatively, glycosphingolipid, which is a white crystalline powder obtained by further purifying the methanol-eluted fraction by preparative liquid chromatography, is more highly active.

  The glycosphingolipid which is this white crystalline powder was β-glucosylceramide.

  Moreover, this invention provides the pharmaceutical containing the above-mentioned iNKT cell activator as another aspect, specifically, an immunopotentiator, an anticancer agent, a cancer metastasis inhibitor, an infection resistance enhancer, It is an allergic agent.

  Specifically, the present invention relates to iNKT cell activation, granulocyte (neutrophil) induction / activation based thereon, macrophage (hereinafter sometimes referred to as “MΦ”) activation action, anticancer agent, cancer metastasis Inhibitors, antiallergic agents, and infection resistance enhancers.

  Furthermore, the present invention, as another aspect, is a diagnostic agent for iNKT cell activation and / or an iNKT cell detection reagent based on the above-mentioned iNKT cell activation action, and also contains an iNKT cell activator. Food and drink.

  According to the present invention, a novel ligand of iNKT cells present in rice bran has been identified, and by clarifying the effectiveness and mechanism of action of this ligand for various pathological conditions, rice having excellent safety as a staple food in Japan. New functional food materials, pharmaceutical raw materials, diagnostic agents, and biochemical research reagents can be provided.

  So far, since no substance that selectively activates iNKT cells in food materials has been found, the present invention is based on the possibility that a novel ligand of iNKT cells exists in rice bran and activation of the cells. The effect of the present invention is particularly remarkable in that it has been found to have an action for improving various pathological conditions.

  In particular, the immunopotentiating effect by selectively activating iNKT cells of the extract containing glycosphingolipids, which is a rice bran extract component clarified by the present invention, recognizes the importance of rice as a staple food by Japanese people. At the same time, the contribution is significant in that they have found new ways to use rice bran, which has been treated as agricultural waste.

It is the figure which showed the chart of Peak 1-5 by HPLC of the methanol recrystallization fraction of Fr.8 of this invention. It is the figure which showed the result of the influence which acts on the NKT cell of the rice bran sphingoglycolipid refined fraction Fr.8. It is the figure which showed the result of the influence which acts on the alpha-CalCer / CD1d tetramer reactive T cell (iNKT cell) of the rice bran sphingoglycolipid refinement | purification fraction Fr.8. It is the figure which showed that iNKT cell activation action was peculiar to rice bran about the influence which the rice bran sphingoglycolipid refinement | purification fraction Fr.8 exerts on NKT cells. It is the figure which showed the result of the influence which acts on the NKT cell of the fraction which removed only Peak 5 among the glycosphingolipid refined fraction Fr.8. It is the figure which showed that the ligand of iNKT cell exists in the fraction containing Peak 5 in Fr.8.

It is the figure which measured the iNKT cell activation effect | action about the recrystallized product of the rice bran sphingoglycolipid refined fraction Fr.8. It is the figure which showed the result of the influence which acts on the tumor metastasis of the rice bran sphingoglycolipid refined fraction Fr.8. It is the photograph which administered the rice bran sphingoglycolipid refined fraction Fr.8 to the melanoma cell transfer mouse | mouth, and showed the result of the disappearance of a tumor cell. It is the figure which showed the result of the influence with respect to the Listeria infection of the rice bran sphingoglycolipid refined fraction Fr.8. It is the figure which showed the expression of the neutrophil count after the administration of rice bran sphingoglycolipid refined fraction Fr.8, and its activation marker. It is the figure which showed the result of the number of granulocytes (neutrophils) in the intrahepatic leukocyte 90 minutes after administration of the rice bran sphingoglycolipid refined fraction Fr.8.

It is the figure which showed the result of the influence on the Listeria exclusion of the neutrophil recognized by the administration of the rice bran sphingoglycolipid refined fraction Fr.8. It is the figure which showed the result of the influence which it has on the microbe killing ability of the rice bran sphingoglycolipid refined fraction Fr.8. It is the figure which showed the result of the influence which acts on MΦ activation of the rice bran sphingoglycolipid refined fraction Fr.8. It is the figure which showed the result of the influence on IFN-gamma production of the rice bran sphingoglycolipid refined fraction Fr.8. It is the figure which showed the result of the influence on NO production of the rice bran sphingoglycolipid refined fraction Fr.8. It is the figure which showed the result of having analyzed rice bran sphingoglycolipid refined fraction Fr.8 with CAPCELL PAK UG120. It is the figure which showed the result of the influence which acts on the NKT cell of Peak 1-7 obtained by analyzing rice bran sphingoglycolipid refined fraction Fr.8 with CAPCELL PAK UG120. It is the figure which showed the result by the mouse | mouth oral administration of the rice bran sphingoglycolipid refined fraction Fr.8.

As described above, the basic aspect of the present invention is rice bran, rice germ and / or an iNKT cell activator derived therefrom, and more specifically, rice bran, rice germ and / or a material derived therefrom. An iNKT cell activator comprising a sphingoglycolipid-purified fraction obtained by fractionating a crude glycosphingolipid-containing fraction obtained by organic solvent extraction by chromatography and / or recrystallization.
Such a glycosphingolipid purified fraction can be obtained specifically by using rice bran or rice germ, and further using these materials, for example, residues obtained in the process of producing rice oil from rice bran or rice germ. Can do.

More specifically, rice bran, rice germ and / or materials derived therefrom are extracted using an organic solvent, particularly alcohol, preferably ethanol, to obtain a crude fraction containing glycosphingolipid.
In the present invention, in the alcohol extraction treatment, as a pretreatment, it is preferable to perform a mixing treatment with, for example, hexane to remove fat-soluble impurities.

A general extraction operation method can be applied to the organic solvent extraction means in the present invention. Moreover, extraction temperature and extraction time cannot be generally limited, and what is necessary is just to carry out about 1 to 24 hours under room temperature-heating.
Specifically, rice bran, rice germ, and / or materials derived therefrom are added with an organic solvent, particularly alcohol, preferably ethanol, and subjected to solvent extraction by appropriate treatment such as stirring. The addition amount of the alcohol is about 0.5 to 10 parts by weight, preferably 1 to 5 parts by weight with respect to 1 part by weight of the raw material. If the amount of alcohol added is small, the extraction operation becomes easy. In short, the amount added should increase the amount of glycosphingolipid extracted.
The extraction temperature is room temperature to about 70 ° C, preferably 30 ° C to 60 ° C. The extraction time is about 1 to 24 hours, preferably about 2 to 5 hours.

  When the alcohol extraction is completed, the alcohol solvent obtained by filtration is distilled off under reduced pressure to obtain a residue. An appropriate amount of acetone is added to the obtained residue and dissolved by heating, and the resulting crude crystals are dissolved again in alcohol. A solution containing an alcohol-solubilized portion is treated with an ion exchange resin, and after distilling off the solvent, an appropriate amount of acetone is treated to obtain a sphingoglycolipid-containing crude extract (that is, a crude fraction).

The purity of the glycosphingolipid in the thus prepared glycosphingolipid-containing crude extract is about 60 to 80%, although it varies depending on the production lot. This crude extract also exhibits iNKT cell activity, granulocyte (neutrophil) induction / activation, and MΦ activation.
The glycosphingolipid purified fraction provided by the present invention is a purified fraction obtained by further fractionating such a glycosphingolipid-containing crude extract, and the fraction is obtained by, for example, processing such as column chromatography. be able to.

Specifically, column chromatography using silica gel is preferable, and examples of the column elution solvent include chloroform, acetone, alcohol, and preferably methanol.
According to the study by the present inventors, it is particularly preferable to elute sequentially with chloroform / chloroform-acetone mixed solution / methanol. By this elution, a glycosphingolipid exhibiting iNKT cell activation action is compared with the methanol elution fraction. It was found to be contained in high purity.
The obtained glycosphingolipid was composed mainly of β-glucosylceramide and composed of glucose, a sphingoid base moiety and a fatty acid residue.

  Furthermore, in order to purify with high purity, it is preferable to concentrate the glycosphingolipid showing iNKT cell activation action with high purity by recrystallizing the methanol elution fraction with methanol.

  Alternatively, the glycosphingolipid exhibiting iNKT cell activation action can be isolated and purified as a single substance by subjecting it to preparative liquid chromatography using a reverse phase column.

As a result of examining the action of the purified glycosphingolipid fraction of the present invention prepared as described above, the obtained glycosphingolipid-containing purified fraction was found to have a strong iNKT cell activating effect. It was confirmed that there was an immune enhancing action through cell activation action, in particular, a cancer metastasis inhibiting action and an infection resistance enhancing action (see Test Examples below).
Therefore, the present invention isolates and identifies a novel ligand of iNKT cell based on the obtained glycosphingolipid-containing purified fraction, and clarifies the effectiveness and action mechanism of this ligand for various disease states. I was able to.

  From this point of view, the present invention provides a new functional food material, biochemical research reagent and pharmaceutical raw material made from rice, which is a staple food in Japan, which is excellent in safety. A wide range of uses can be expected for the purified glycosphingolipid fraction.

  Currently, many people are health-conscious, and the health food boom is overheating, and people's interest in anti-aging (anti-aging / anti-aging) has increased. Studies have revealed that immune system abnormalities are closely related to aging. Therefore, there is a strong social demand for the development of highly safe health foods, health drinks, cosmetics, and in particular, foods, drinks, and cosmetics that particularly enhance immunity.

  In addition to the existing diseases, new diseases appear, and allergies such as cancer, emerging infections, re-emerging infectious diseases, and hay fever are increasing, and immediate measures against these diseases are socially necessary. It is said that. It can be said that the present invention has a very high possibility of contributing to overcoming the above-described problems.

So far, no substance that selectively activates iNKT cells in food has been found. Therefore, the present invention is based on the possibility that novel ligands of iNKT cells exist in rice bran and various types of activation through activation of the cells. It is very specific in that it has been found to have an effect of improving the pathological condition.
The immunostimulatory effect of the glycosphingolipid fraction (new iNKT cell ligand) extracted and fractionated from rice bran, which was revealed this time, reaffirms the importance of rice as a staple food for Japanese people, and many are discarded for agriculture. In terms of finding a new method for using rice bran that has been processed as food, the contribution is significant.

  Recently, chemically synthesized α-GalCer (originally found in sponges) is a ligand for iNKT cells and has been shown to exhibit a strong antitumor effect and infection control action depending on iNKT cells. Therefore (Non-patent Documents 1 and 2), anticipation as anticancer agents, antiinfectives, and antiallergic agents is increasing. However, since mammals, including humans, are unlikely to encounter sponges, many researchers have since started searching for natural ligands. As a result, α-glucuronosylceramide (Non-patent Documents 3 to 6) present in the cell walls of Sphingomonas / Ehrlichia, α-galactosyldiacylglycerol (Non-patent Document 7) present in Borrelia are natural ligands of the cells. (It has been reported that isoglobotrihexosylceramide present in lysosomes is also a natural ligand of this cell (Non-patent Documents 8 and 9), but is currently negative (Non-patent Document 10). )).

  At present, many researchers are searching for ligands of iNKT cells using chemically synthesized glycosphingolipids, but none of them has a strong activity like α-GalCer. Since it is unclear to date whether natural ligands are present or not, the novel iNKT cell natural ligands found in the rice bran-derived purified glycosphingolipids that have been clarified this time are It can be said that it shows a great contribution in this field.

  INKT cell activator comprising the purified glycosphingolipid fraction, anticancer (cancer metastasis inhibitor), infection resistance enhancer, antiallergic agent, granulocyte (neutrophil) induction For the activator, macrophage activator, and research reagent, this fraction can be used as it is or mixed with a pharmacologically acceptable carrier according to known means generally used in the preparation of pharmaceutical preparations. Thus, for example, it can be safely administered as a pharmaceutical preparation such as a tablet, powder, granule, capsule, liquid, injection, suppository, sustained-release agent and the like.

Specifically, the content of the purified glycosphingolipid fraction may be 1 to 100% by weight of the entire preparation, but is preferably 10 to 100% by weight, and more preferably 50 to 100% by weight.
The dose of the purified glycosphingolipid fraction is not particularly limited because it varies depending on the administration subject, target organ, symptom, administration method and the like, but generally 0 per day for a patient (with a body weight of 60 kg). .1 to 100 mg, preferably 1.0 to 50 mg, more preferably about 1.0 to 20 mg.
The administration method of the present glycosphingolipid purified fraction is not particularly limited and can be appropriately selected. Of these, intravenous injection is preferable. However, it goes without saying that it may be administered locally in a solid tumor.

Examples of pharmacologically acceptable carriers include excipients, lubricants, binders and disintegrants in solid formulations, or solvents, solubilizers, suspending agents, isotonic agents, buffers in liquid formulations. And soothing agents. Further, if necessary, additives such as conventional preservatives, antioxidants, colorants, sweeteners, adsorbents, wetting agents and the like can be used in appropriate amounts.
Examples of the excipient include lactose, sucrose, D-mannitol, starch, corn starch, crystalline cellulose, light anhydrous silicic acid and the like. Examples of the lubricant include magnesium stearate, calcium stearate, talc, colloidal silica and the like. Examples of the binder include crystalline cellulose, sucrose, D-mannitol, dextrin, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, starch, sucrose, gelatin, methylcellulose, sodium carboxymethylcellulose and the like.

  Examples of the disintegrant include starch, carboxymethyl cellulose, carboxymethyl cellulose calcium, carboxymethyl starch sodium, L-hydroxypropyl cellulose, and the like. Examples of the solvent include water for injection, alcohol, propylene glycol, macrogol, sesame oil, corn oil, olive oil and the like. Examples of the solubilizer include polyethylene glycol, propylene glycol, D-mannitol, benzyl benzoate, ethanol, trisaminomethane, cholesterol, triethanolamine, sodium carbonate, sodium citrate and the like.

  Examples of the suspending agent include surfactants such as polysorbate, stearyltriethanolamine, sodium lauryl sulfate, laurylaminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride, and glyceryl monostearate; for example, polyvinyl alcohol, polyvinyl Examples thereof include hydrophilic polymers such as pyrrolidone, sodium carboxymethylcellulose, methylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, and hydroxypropylcellulose.

  Examples of the isotonic agent include glucose, D-sorbitol, sodium chloride, glycerin, D-mannitol and the like. Examples of the buffer include buffer solutions of phosphate, acetate, carbonate, citrate and the like. Examples of soothing agents include benzyl alcohol. Examples of the preservative include parahydroxybenzoic acid esters, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, sorbic acid and the like. Examples of the antioxidant include sulfite, ascorbic acid, α-tocopherol and the like.

  The glycosphingolipid obtained by this method may be chemically synthesized, but is preferably obtained from rice bran, rice germ and / or a material derived from these from the viewpoint of safety.

  It should be noted that the iNKT cell activator comprising the purified glycosphingolipid fraction of the present invention, an anticancer (cancer metastasis suppressant) agent containing the activator, an infection resistance enhancer, an antiallergic agent, a granulocyte (neutral cell) Sphere) inducer / activator, macrophage activator, and research reagent mean a drug used for the prevention or treatment of the diseases described above based on the therapeutic or preventive effect of the purified glycosphingolipid fraction However, a drug containing the purified glycosphingolipid fraction together with other active ingredients as an adjuvant or the like is not meant.

  Hereinafter, the present invention will be described in detail by describing the actual isolation of the rice sphingoglycolipid and the action of the purified glycosphingolipid purified fraction. It is not limited.

Test Example 1: Isolation of rice bran glycosphingoglycolipid A hydrated gum obtained by adding water to rice crude oil was degreased and neutralized, and then insolubles were removed to obtain a raw material containing about 1% glycosphingolipid.
500 g of this raw material was dissolved by heating in an appropriate amount of hexane to remove fat-soluble impurities. To the residue obtained by distilling off the solvent, 750 mL of ethanol was added and stirred to remove the solvent. An appropriate amount of acetone was added to the obtained residue, dissolved by heating, and recrystallized. The obtained crude crystals were redissolved in ethanol to remove glycosides, and then the ethanol soluble fraction was separated from an anion exchange resin (Amberlite IRA-900: manufactured by Organo) and a cation exchange resin (Amberlite). 200-C: manufactured by Organo Corporation). The solvent of the resin-treated product was removed, and the residue was recrystallized with an appropriate amount of acetone to isolate 1.0 g (yield 0.5%) of rice bran sphingoglycolipid. The purity of the glycosphingolipid thus obtained was quantified by ELSD-HPLC (liquid chromatography with a light scattering detector).
By the same method, glycosphingolipids could be obtained from corn, straw, wheat, pig brain and the like.

Test Example 2: Isolation of rice bran sphingoglycolipid 8 g of rice bran sphingoglycolipid obtained in Test Example 1 above was dissolved in chloroform, and silica gel column chromatography (40 g of silica gel for flash chromatography was suspended in chloroform: spherical, Kanto Chemical) Fractionation).
Fractions were eluted with 600 mL of chloroform: acetone = 10: 0 → 9: 1 → 8: 2 → 7: 3 → 6: 4 → 5: 5 → 4: 6 and 600 mL of methanol to remove the solvent. .
The collection examples of the fractions thus obtained were as shown in Table 1 below.

  From the results of gas chromatography (GC), thin layer chromatography (TLC: silica gel, chloroform: methanol = 10: 1, colored with concentrated sulfuric acid) and liquid chromatography of each fraction, fractions 5 and 6 (Fr. And Fr.6) were glycosides, and fractions 7 and 8 (Fr.7 and Fr.8) were glycosphingolipid purified fractions mainly composed of glycosphingolipids.

Fraction 8 (Fr. 8) described above uses an ODS-120A (4.6 mm (ID) 15.0 cm) column, and the conditions are 40 ° C. with methanol / water = 15/1 (flow rate: 1.00 mL / min) as the eluting solvent. As a result of performing HPLC analysis below, it was composed of at least five peaks (Peak 1 to 5), and recrystallization with methanol revealed that Peak 5 was concentrated.
The peak state on the HPLC is shown in FIG.

  Further, when the glycosphingolipid fraction purified using another extraction / separation / purification method was analyzed by HPLC, the fraction lacking only Peak 5 described above was also successfully purified.

  When the purified glycosphingolipid fraction extracted and separated from corn, straw, wheat, and pig brain was analyzed by the same method, Peak 5 described above was not observed.

The pharmacological action of the glycosphingolipid purified fraction obtained as described above was examined.
The safety (cell proliferation ability and cell viability) for the living body has been confirmed in the safety test.

Pharmacological test example:
[Use mouse]
C57BL / 6 mice and various iNKT cell-deficient mice (all female mice 8-12 weeks old) were subjected to the experiment.

[Used tumor strain]
Melanoma cell line (B16) and T lymphoma (EL4) were subjected to the experiment.

[Used strain]
Listeria monocytogenes (EGD strain) (Listeria) and Salmonella enterica Serovar Typhimurium (LT-2 strain) were used for the experiment.

[Administration]
150 μg of each fraction was administered intraperitoneally to C57BL / 6 mice and various iNKT cell-deficient mice.
In the following experiment, mice having a weight difference of less than 1 g between groups were used.

[antibody]
Monoclonal antibody (mAb) against Fcγreceptor (R) (2.4G2) and Ly6G (Gr-1: RB6-8C5) was purified from the hybridoma culture supernatant, or fluorescein isothiocyanate (FITC) -labeled anti-CD3ε mAb (145-2C11), FITC-labeled anti-TCR-β mAb (H57-597), Phycoerythrin (PE) -labeled anti-CD11b mAb (Mac-1: M1 / 70), FITC-labeled anti-Ly6G mAb (Gr-1: REB-8C5) ) And biotin-labeled anti-NK1.1 mAb (PK136) purchased from BD PharMingen (Hamburg, Germany) were subjected to the experiment.

[reagent]
Fetal calf serum (FCS) is Trace Biosciences (Castle Hill, HSW, Australia), Percoll (specific gravity: 1.124 g / mL) is Biochrom (Berlin, Germany), RPMI 1640 is Nissui Pharmaceutical Co., Ltd. (Tokyo, Japan) Japan), HEPES is Dojindo Laboratories (Kumamoto, Japan), L-glutamine and penicillin / streptomycin are Invitrogen (Carlsbad, CA, USA), 2-mercaptoethanol, sodium bicarbonate, ammonium chloride, TRIS (2-amino-) 2-hydroxymethyl-1,3-propanediol) and sodium azide are Wako Pure Chemical Industries, Ltd. (Osaka, Japan), bovine serum albumin (BSA) is Thermo (Hamilton, New Zealand), streptavidin (Streptavidin: SA) labeled Cy5 was purchased from BD PharMingen (Hamburg, Germany), and paraformaldehyde purchased from Merck (Darmstadt, Germany) was used for the experiment.

[Production of α-GalCer / CD1d tetramer]
Mouse CD1d / β2-microglobulin (β2m) tetramer was prepared using the BirA biotinylation site and a baculovirus expression system using CD1d. That is, an Sf9 insect cell line (BD Bioscience, Heidelberg, Germany) was infected with a virus expressing mouse CD1d / β2m and cultured in an Sf-900 serum-free medium. Culture supernatants 3 or 4 days after infection were collected and concentrated with a hollow fiber tangential flow module (MiniKros 1100 cm ² , Spectrum, MembraPure, Boddenheim, Germany). CD1d molecules were purified by metal chelate chromatography (Chelating sepharose Fast flow, Amersham Pharmacia Biotech) using a cobalt chloride ion charged NTA-Sepharose column (Amersham Pharmacia Biotech, Uppsala, Sweden). The protein is eluted with 200 mM imidazole and concentrated to 0.5 mL by ultrafiltration (Ultrafree Units, Millipore, Bedford, MA). The accuracy and protein amount are confirmed by SDS-PAGE and Bradford reagent (BioRad, Munich, Germany), respectively. did. The purified CD1d protein was conjugated with biotin with BirA enzyme and purified by gel filtration (Superdex 200 HR10 / 30, Amersham Pharmacia Biotech). Α-GalCer (Kirin Pharma, Co., LTD., Dissolved in 0.5% Tween 20-added phosphate-buffered saline (PBS) so that the molarity is 3: 1 (lipid: protein). Tokyo, Japan) and CD1d / β2m protein were mixed and allowed to react overnight at room temperature. SA-PE was added to α-GalCer / CD1d / β2m monomer at a molar concentration of 1: 4 (monomer: SA-PE) to form a tetramer, and gel filtration (Superdex 200 HR10 / 30, PE-labeled α-GalCer / CD1d / β2m tetramer bound to α-GalCer was purified by Amersham Pharmacia Biotech.

[Preparation of intrahepatic leukocytes]
C57BL / 6 mice were euthanized with cervical dislocation or diethyl ether, complete medium (CM: 20 mM HEPES / 10% sodium bicarbonate / L-glutamine (293 μg / mL) / penicillin (100 U / mL) / streptomycin (100 μg) / mL) / 10% FCS / 2-mercaptoethanol (10 ⁵ M) -containing RPMI 1640 medium), and then the liver was extracted. The liver was crushed with a metal mesh, and the cell suspension was centrifuged at 500 rpm for 20 seconds. The supernatant was centrifuged at 1,500 rpm for 5 minutes, 40% percoll (diluted with CM) was added to the sediment, and the mixture was overlaid with 70% percoll (diluted with CM). After centrifuging at 1,800 rpm for 23 minutes, an intermediate layer of 40% and 70% percoll was recovered. Hemolysis with hemolysis buffer solution (dissolved 8.3 mg of ammonium chloride in distilled water and 111 mL of 0.17 M Tris buffer (pH 7.6)), then washed with CM to 1 × 10 ⁶ / mL The cells were suspended in CM.

[Preparation of intrahepatic granulocytes (neutrophils)]
After C57BL / 6 mice were euthanized by cervical dislocation, the liver was perfused with CM and the liver was removed. After crushing with a metal mesh, the cell suspension was layered on top of the 1.085 g / mL percoll solution having a specific gravity of 1.095 g / mL and the 1.085 g / mL percoll solution on top of each other. After centrifugation at 500 g for 4 minutes at 4 ° C., the intermediate layers of 1.085 g / mL and 1.095 g / mL Percoll solution were collected, washed, and suspended in a phenol red-free MEM medium containing HEPES and 10% FCS. 95% or more of granulocytes (neutrophils) are obtained by this method, and it is confirmed by staining with anti-Ly6G mAb that 94 to 96% of granulocytes (neutrophils) are present in this fraction. It was done.

[Preparation of intraperitoneal cells and peritoneal MΦ]
After C57BL / 6 mice were euthanized with diethyl ether, 5 mL of CM was administered intraperitoneally to collect intraperitoneal cells. After washing, the cells were cultured in a 6-well tissue culture plate (Corning Inc., Canton, NY) at 37 ° C. for 2 hours, and adherent cells and non-adherent cells were collected separately. In addition, it was confirmed by flow cytometry and immunohistochemical staining that 95% or more of the adherent cells obtained by this method were MΦ.

[Flow cytometry]
In order to prevent non-specific binding of the antibody, the cells were reacted with anti-FcγR mAb at 4 ° C. for 15 minutes, and then reacted with FITC, PE and biotin-labeled mAb at 4 ° C. for 15 minutes. Biotin-labeled antibodies were visualized with SA-label 5 (BD ParMingen). After staining, the cells were fixed with 1% paraformaldehyde (PFA) -containing PBS (−), taken up with a flow cytometer (FACSCalibur (BD Biosciences)), and analyzed with CellQuest software (BD Biosciences). For staining with α-CalCer / CD1d tetramer, after blocking, it was reacted with PE-labeled α-CalCer / CD1d tetramer at room temperature for 15 minutes.

[Dynamic analysis of iNKT cells]
After preparing leukocytes as described above from the liver of C57BL / 6 mice that have been pre-purified with rice bran-derived glycosphingolipid or C57BL / 6 mice that have been intraperitoneally administered with vehicle, iNKT cell dynamics are analyzed by flow cytometry (mAb · α-GalCer / CD1d tetramer). Both).

[Dynamic analysis of granulocytes (neutrophils)]
Leukocytes were prepared as described above from the liver of C57BL / 6 mice that had been pre-purified with a rice bran-derived glycosphingolipid or a vehicle intraperitoneally, and the dynamics of granulocytes (neutrophils) were analyzed by flow cytometry.

[Tumor metastasis suppression experiment]
C57BL / 6 mice that have been intraperitoneally administered with a purified fraction of glycosphingolipids derived from rice bran or intraperitoneally inoculated with melanoma cells (B16) or T Lymphoma (EL4). First, the degree of metastasis in the lung on the 40th day after inoculation was visually observed.
In addition, the purified rice sphingoglycolipid fraction was administered to mice 25 days after inoculation with B16, and the degree of metastasis in the lungs 60 days after administration was visually observed.

[Infection experiment]
C57BL / 6 mice that were intraperitoneally administered with a purified fraction of glycosphingolipids derived from rice bran or intraperitoneally in advance were infected with Listeria, and the spleen of mice on the fourth day of infection was visually observed, and live in the liver and spleen The number of bacteria was measured.

[Granulocyte (neutrophil) disappearance experiment]
Mice were given 150 μg of anti-Ly6G mAb intraperitoneally one day before Listeria infection. The disappearance of 95% or more of Ly6G ⁺ cells was confirmed by flow cytometry and immunohistochemical staining using fluorescently labeled anti-Ly6G mAb. In the group that received anti-Ly6G mAb, Ly6G ⁺ cells could not be detected by fluorescently labeled rat IgG2b (secondary antibody against anti-Ly6G mAb) (the antibody was not bound to the cells, but the cells were completely lost) Confirmed).

[Measurement of respiratory burst of granulocytes (neutrophils)]
As described above, granulocytes (neutrophils) were purified from the liver, and respiratory burst was measured by luminol-dependent chemiluminescence (CL). That is, 200 μL of granulocyte (neutrophil) suspension (5 × 10 ⁵ cells / mL) was mixed with 50 μL of luminol solution (1.0 × 10 ⁻⁶ M; Laboscience Co., Tokyo, Japan) 37 The reaction was carried out at 5 ° C. for 5 minutes. After adding 200 μL of opsonized Listeria (10 ⁷ CFU / mL) or 10 ^-6 M N-formyl-methionyl-leucyl-phenylalanin (FMLP; Sigma-Aldrich, St. Louis, MO), maintain at 37 ° C The measurement was performed for 15 minutes with a luminometer (Lumiphotometer TD-4000, Laboscience).

[Measurement of bactericidal ability of MΦ]
Adherent cells (MΦ) purified from the abdominal cavity of mice (1 × 10 ⁻⁶ / well) were cultured in 24-well tissue culture plates (Corning) for 2 hours, and then listeria (1 × 10 ⁷ / well) (MOI : 10) infected. After reacting for 60 minutes, cells that had not been incorporated into MΦ were removed with RPMI 1640 containing 10% FCS and gentamicin (Sigma-Aldrich, Tokyo, Japan) (5 μg / mL) in which cells were pre-warmed ( P ₆₀ ). The cells were further incubated for 30 minutes in RPMI 1640 containing 10% FCS and gentamicin (5 μg / mL) (B ₃₀ ). The cells were treated with 0.5% saponin (Sigma-Aldrich), sonicated, seeded on Tryptic soy agar plates, and the viable count (Colony-forming unit: CFU) was measured 48 hours later. The bactericidal ability was determined by the following calculation formula.
100 − {(B ₃₀ / P ₆₀ ) × 100} (%)
Data was calculated by the following formula and expressed as a Bactericidal index.
Treatment group _{{100 - ((B 30 /} P 60) × 100)} / untreated group _{{100 - ((B 30 /} P 60) × 100)}

[Measurement of IFN-γ and IL-4]
Production of IFN-γ and IL-4 was measured using Quantikine (registered trademark / R & D Systems Inc., Minneapolis, Minn.). The detection limit was 2 pg / mL.

[Measurement of Nitric oxide (NO)]
NO was measured by Griess Reagent System (registered trademark / Promega Co., WI). The detection limit was 2.5 μM.

[Measurement of Respiratory burst of MΦ]
Respiratory burst of peritoneal MΦ was measured by CL. That is, the addition of 30μL of luminol solution (1.0 × 10 ^-6 M) in 400μL of peritoneal MΦ suspension (1.2~1.5x10 ⁶ cells), gentle stirring 37 ° C., and allowed to react for 5 minutes. Thereafter, opsonized Listeria (10 ⁸ CFU / 150 μL) was added, and light release was allowed to react with Lumiphotometer TD-4000 (Laboscience) for 15 minutes, and the peak was measured.

[Measurement of iNKT cell activation by oral administration of mice]
C57BL / 6 mice were used, and Fr. 8 was made into 100 nm nano-dispersed powder, and 500 μL of the 200 mg / mL nano-dispersed liquid (corresponding to 100 mg / mouse) was continuously administered for 3 days using an oral sonde. Later hepatic leukocytes were analyzed by flow cytometry.

[Results and discussion]
1. Effect of purified rice bran glycosphingoglycolipid fraction on intrahepatic leukocytes Mice were administered purified fraction of rice glycosphingoglycolipid or vehicle, and 24 hours later, leukocytes present in the liver were analyzed by flow cytometry.
In the vehicle administration group, almost no change was observed in each cell population. When each fraction obtained by fractionating crude rice sphingoglycolipids was administered, no significant change was observed when fractions other than Fr.8 were administered, but in the Fr.8 administration group , NK (CD3 ^- NK1.1 ⁺⁾ cells, and B (CD3 ^- NK1.1 ^-) but cells were little affected, NKT (CD3 ⁺ NK1.1 ⁺⁾ cells selectively remarkable Hesi, Along with this, an increase in T cells was observed (the results are shown in FIG. 2). Among the unpurified glycosphingolipids obtained in Example 1, the same phenomenon was observed in lots containing Peak 5, although there were some differences.
NKT cells are composed of several subpopulations. Among them, α-GalCer / CD1d tetramer is detected that only iNKT cells expressing Vα14 / Jα18 as T cell receptors (TCR) cannot be selectively detected. (The results are shown in FIG. 3).

When the presence / absence and strength of glycosphingolipids similarly extracted from corn, straw, wheat, and pig brain were examined by the above-described method, iNKT cells were not lost at all.
That is, it was verified that the detected activity was unique to rice bran (the result is shown in FIG. 4).

As described above, Fr. 8 is composed of at least five peaks (Peak 1 to 5). When the activity of the fraction lacking Peak 5 was examined by the method described above, this fraction contained iNKT cell activity. No conversion effect (disappearance of iNKT cells) was observed (the results are shown in FIG. 5).
In addition, when the effect of a fraction rich in Peak 5 on iNKT cells was examined by repeating recrystallization, iNKT cell activation action (disappearance of iNKT cells) was observed in this fraction, that is, the recrystallization fraction. (The results are shown in FIG. 6).
From this, it was determined that the ligand of iNKT cells was present in the fraction containing Peak 5 in Fr.

For recrystallization, 9.13 g of Fr. 8 was dissolved in 50 mL of methanol and left overnight at 4 ° C. to obtain 3.80 g of crystals (Ppt 1), and 2.08 g of the obtained crystals (Ppt 1). It was dissolved in 150 mL of methanol and left overnight at 4 ° C. to obtain 0.83 g of crystals (Ppt 2).
On the other hand, the mother liquor from which the first crystals were obtained was concentrated to dryness to obtain 5.45 g of solid (Sup. 1), and the mother liquor from the second recrystallization was concentrated to dryness to obtain 1.2 solids (Sup 2).
Each of the primary crystal (Ppt 1), secondary crystal (Ppt 2), primary mother liquor solid (Sup 1) and secondary mother liquor solid (Sup 2) obtained by this recrystallization operation is activated by iNKT cell (iNKT cell activation). (Disappearance) in comparison with Fr.8 before recrystallization, iNKT cell activation action (disappearance of iNKT cells) was confirmed in all samples, especially in the recrystallized sample. It was.
Therefore, it was confirmed that the recrystallized product of Fr. 8 has a stronger iNKT cell activation effect (the result is shown in FIG. 7).

2. Effect of purified rice spruce glycosphingolipid fraction on tumor metastasis Mice previously administered with rice spruce glycosphingolipid fraction or vehicle were inoculated with melanoma cells (B16) intravenously, and the survival rate and lung The condition was observed.
In the untreated group and the vehicle-treated group, B16 metastasis to the lung (lung of the lung) was observed and death was observed from an early stage, but in the Fr. 8 administration group, no metastasis to the lung was observed. All mice survived even when half a year had passed (the results are shown in FIG. 8, where * is p <0.01).
In iNKT cell-deficient mice, even if the purified rice sphingoglycolipid fraction was administered, little effect was observed. Similar phenomenon was observed when EL4 was used.
In addition, when B16 was inoculated into mice and Fr. 8 was administered to mice in which B16 had spread to the lung, B16 was completely eliminated from the lung. That is, when mice were inoculated with B16, metastasis to the lung was already observed on the 25th day after inoculation, but when Fr. 8 was administered at that time, B16 metastasized to the lung (pulmonary blackening) was completely It disappeared (the result is shown in FIG. 9).

3. Effect of purified rice bran glycosphingolipids on infection protection Infect mice with Listeria sphingoglycolipid purified fraction or Vehicle in advance and infecting Listeria via vein, and visually observing the spleen after infection and survival rate In addition, the number of viable bacteria in the liver and spleen on the fourth day of infection was measured.
In the untreated group and the vehicle-treated group, strong inflammation was observed in the spleen after infection (salami-like), but when each fraction obtained by fractionating a rice bran sphingoglycolipid crude product was administered, When fractions other than Fr. 8 were administered, no significant change was observed. On the other hand, no inflammatory image was observed in the spleen of the Fr.8 treatment group, which was almost the same as before infection.
In the Fr. 8 treatment group, the number of viable bacteria in the liver and spleen was significantly suppressed in the Fr. 8 treatment group (FIG. 10 shows the result of the number of viable bacteria in the liver. In the figure, * indicates p < 0.05: Fr.8 vs. Vehicle).

4). Effects of purified rice sphingoglycolipid fraction on neutrophil induction
C57BL / 6 mice were treated with purified rice sphingoglycolipid fraction or Vehicle, and granulocytes (neutrophils) present or accumulated in the liver 24 hours later were analyzed by flow cytometry. In the untreated or vehicle-treated groups, almost no cells (granulocytes (neutrophils)) showing high values in SSC were detected. When each fraction obtained by fractionating crude rice sphingoglycolipid was administered, no significant change was observed when fractions other than Fr.8 were administered, but the group administered with Fr.8 In SSC, many cells (granulocytes (neutrophils)) showing high values were detected.
Usually, granulocytes express Ly6G and CD11b at a high frequency. Therefore, when these cells were analyzed using fluorescently labeled anti-Ly6G mAb and anti-CD11b mAb, most of them were Ly6G ⁺ CD11b ⁺ (granulocytes (preferably (The results are shown in FIG. 11. In the figure, * and ** are p <0.01: Fr. 8 vs. Vehicle).
In addition, when the dynamics of granulocytes (neutrophils) were examined in more detail, granulocytes were already 90 minutes later after administration of Fr. 8 among the fractions obtained by fractionating the rice bran glycosphingoglycolipid crude product. (Neutrophils) A marked increase was observed, accounting for nearly 50% of leukocytes in the liver, and its effect was much stronger than α-GalCer (the results are shown in FIG. 12). )

5). Effects of administration of the purified rice sphingoglycolipid fraction on neutrophil listeria elimination In order to eliminate granulocytes (neutrophils) from the body, C57BL / 6 mice were administered anti-Ly6G mAb in advance. Then, how resistance to Listeria infection changes was analyzed by measuring CFU in the liver on the fourth day of infection.
CFU in the liver of the PBS-treated group increased due to disappearance of granulocytes (neutrophils), but the degree of increase was significantly higher in the Fr. 8 administration group (the results are shown in FIG. 13). In the figure, * indicates p <0.05: anti-Ly6G mAb vs. PBS; ** indicates p <0.05: Fr. 8 vs. PBS; *** indicates p <0.01: Fr. 8 vs. Fr. 8 + anti Ly6G mAb).

6). Effect of purified rice sphingoglycolipid fraction on neutrophil activation Since Listeria is an intracellular parasitic bacterium, it can survive and proliferate in phagocytic cells, but most of them are granulocytes (neutrophils) Sterilized and processed by a ball). Sterilization in granulocytes (neutrophils) is mainly due to respiratory (Oxidative) burst, so CL when stimulating granulocytes (neutrophils) present or accumulated in the liver with FMLP or opsonized Listeria Responses were compared between the Vehicle treatment group and the Fr.8 treatment group. When stimulated with FMLP and opsonized Listeria, CL responsiveness increased in the group administered Fr.8 (Table 2 below).

a: p <0.05: Fr. 8 vs. Vehicle
b: p <0.01: Opsonized Listeria, FMLP vs. PBS
c: p <0.005: Opsonized Listeria, FMLP vs. PBS
d: p <0.05: opsonized Listeria vs. FMLP

  As shown in the table, also in the vehicle-treated group, when stimulated with FMLP and opsonized Listeria, CL responsiveness increased, but the degree of increase was higher than that of the group that had been administered Fr. The value was significantly low. In addition, when Fr. 8 was administered, the respiratory (Oxydative) burst of granulocytes was higher for pathogens than for soluble stimulants. On the other hand, in mice lacking iNKT cells, no increase in Respiratory (Oxydative) burst was observed even when Fr. 8 was administered.

7). Effect of MΦ on the bactericidal ability of purified rice sphingoglycolipid fraction In addition to granulocytes (neutrophils), MΦ is essential for eliminating Listeria. The effect of was examined. MΦ prepared from the abdominal cavity of C57BL / 6 mice administered with Fr. 8 or Vehicle was infected with Listeria in vitro, and the degree of sterilization at a certain time was measured. That is, after phagocytosing Listeria in MΦ for 60 minutes, bacteria that were not phagocytosed were removed by washing, and how much bacteria were sterilized in 30 minutes was analyzed. The bactericidal ability of MΦ collected from mice administered with Fr.8 showed higher bactericidal ability than MΦ collected from untreated or vehicle-treated mice (the results are shown in FIG. 14). Is p <0.05: Fr.8 vs. Vehicle, Nil).
Although the bactericidal ability slightly increased by the vehicle treatment, the degree was clearly lower than that of MΦ recovered from the mice administered with Fr.
On the other hand, in mice lacking iNKT cells, no increase in bactericidal activity was observed even when Fr. 8 was administered.

8). Effect of MΦ on the phagocytic ability of purified rice sphingoglycolipid fraction
The number of Listeria infected with Listeria was infected with MΦ purified from the abdominal cavity of Fr.8 or Vehicle-administered mice, and phagocytosed in MΦ was compared by immunohistochemical method using anti-Listeria Ab labeled with fluorescence. The number of Listeria phagocytosed in MΦ was significantly higher in the Fr. 8 treatment group than in the Vehicle or untreated group.
On the other hand, in mice lacking iNKT cells, no increase in phagocytic ability was observed even when Fr. 8 was administered.

9. Effect of purified rice sphingoglycolipid fraction on MΦ activation
In order to clarify whether or not humoral factors produced from iNKT cells are involved in the activation of MΦ by Fr.8, MΦ purified from the abdominal cavity of C57BL / 6 mice was preliminarily used as Fr.8 or Vehicle cultured in the presence / absence of intraperitoneal fluid prepared from the peritoneal cavity of treated mice, and compared by the same method as described above. When the intraperitoneal fluid prepared from the intraperitoneal cavity of the untreated or vehicle-treated group was treated, no increase in the bactericidal ability of MΦ against Listeria was observed, but the abdominal cavity prepared from the abdominal cavity of the Fr.8-treated group. When the internal solution was treated, the bactericidal ability of MΦ against Listeria was significantly enhanced (the results are shown in FIG. 15. In the figure, * is p <0.05: Fr.8 vs. Vehicle, Nil) .
On the other hand, no increase in bactericidal activity was observed even when MΦ was co-cultured with intraperitoneal fluid prepared from mice lacking iNKT cells administered with Fr.

10. Effect of purified rice sphingoglycolipid fraction on IFN-γ production
Since IFN-γ strongly activates MΦ and is essential for protection against Listeria infection, the ability of Fr.8 to induce IFN-γ production was examined.
Although no IFN-γ was detected in the intraperitoneal fluid of the untreated group, a large amount of IFN-γ was detected in the intraperitoneal fluid of the group treated with Fr. 8 (the results are shown in FIG. 16). In the figure, * indicates p <0.05: Vehicle vs. Nil; ** indicates p <0.01: Fr. 8 vs. Vehicle; + indicates p <0.005: Fr. 8 vs. Nil).
Although production of IFN-γ was also confirmed slightly in the intraperitoneal fluid of the vehicle-treated group, it was clearly lower than that of the group treated with the purified rice sphingoglycolipid fraction.

11. Effects of purified rice sphingoglycolipid fraction on NO production NO is not only produced from MΦ after Listeria infection, but also plays an important role in the sterilization of Listeria, and NO production is induced by IFN-γ Therefore, the influence of Fr. 8 on NO production of MΦ was analyzed.
MΦ prepared from the abdominal cavity of C57BL / 6 mice previously administered with Fr. 8 or Vehicle was cultured in vitro, and the amount of NO in the culture supernatant was compared.
No NO was detected in the untreated group, but a large amount of NO was detected in the Fr.8 treated group (the results are shown in FIG. 17. In the figure, * indicates p <0.05: Vehicle vs. Nil; ** is p <0.01: Fr.8 vs. Vehicle; + is p <0.005: Fr.8 vs. Nil).
Although some NO production was also confirmed in the vehicle-treated group, the value was clearly lower than that in the group treated with the purified rice sphingoglycolipid fraction.

  As described above, the iNKT cell activator provided by the present invention comprises a purified fraction of glycosphingolipid obtained from a plant material such as rice bran and rice germ sugar, and is highly safe and immune. It is understood that it has an enhancing action.

In the separation of the rice bran glycosphingolipid of Test Example 2 described above, it was fractionated into Fr. 1 to 8 fractions by silica gel column chromatography, and the rice bran glycosphingoglycolipid was concentrated in the Fr. It was found to be contained.
The Fr. 8 fraction uses an ODS-120A (4.6 mm (ID) 15.0 cm) column and is 40 ° C. with methanol / water = 15/1 (flow rate: 1.00 mL / min) as the eluting solvent. As a result of performing the HPLC analysis below, it was composed of at least 5 peaks (Peak 1 to 5).

  Therefore, the Fr. 8 fraction was subjected to HPLC analysis under different conditions, and the separation of rice bran sphingoglycolipid was examined.

CAPCELL PAK UG120 (Shiseido) 4.6Φ × 250mm was used as the column, and analysis was performed at 40 ° C. with elution solvent: methanol / water = 95/5, flow rate: 1.00 mL / min, and detection wavelength: 220 nm. As a result, it was found that it was composed of at least 7 peaks (Peak 1 to 7).
The peak state on the HPLC is shown in FIG.

The effect of each peak (Peak 1-7) on the fraction iNKT cells was examined.
100 μg of each peak was administered intraperitoneally to C57BL / 6 mice, and intrahepatic leukocytes 24 hours later were analyzed by flow cytometry. In the Vehicle treatment group, α-GalCer / CD1d tetramer-reactive cells were detected at high frequency, but in the livers of mice treated with Peaks 1, 2, 6 and 7, the cells were almost undetectable. In addition, although not completely in the groups administered Peak 4 and 5, disappearance of α-GalCer / CD1d tetramer reactive cells was observed. Since disappearance of iNKT cells is observed only when ligands are bound to iNKT cells, these results show that all peaks except Peak 3 have the degree of iNKT cell ligands to varying degrees. It suggests. The analysis was performed using 2 mice in each group, and the same results were obtained, so representative results were shown.
The results are shown in FIG.

12 INKT cell activation by oral administration in mice
Using C57BL / 6 mice, Fr. 8 was made into a nano-dispersion of about 100 nm by a wet method, and 100 µg equivalent / mouse of the nano-dispersion was administered continuously for 3 days using an oral sonde. Intrahepatic leukocytes were analyzed by flow cytometry. Two samples of nano-dispersions of Lot.119 and Lot.124 were used as administration samples.
As a result, CD3 + NK1.1 + cells were frequently detected in the vehicle-treated group, but in the livers of mice administered with dispersions of Lot. Was recognized.
Since most of the CD3 + NK1.1 + cells are iNKT cells and the disappearance of the CD3 + NK1.1 + cells is observed only when the ligand is bound to the iNKT cells, these results indicate that the sphingo of the present invention. This suggests that the glycolipid fraction (Fr. 8) is not only absorbed from the intestinal tract, but also the glycosphingolipid absorbed from the intestinal tract has entered the liver and bound to iNKT cells.
The analysis was performed using two mice in each group, and exactly the same results were obtained, so representative results are shown in FIG.

According to the present invention, a novel ligand of iNKT cells is isolated and identified in rice bran, and by clarifying the effectiveness and mechanism of action of this ligand for various pathologies, rice having excellent safety as a staple food in Japan. It is possible to provide a new functional food material and a raw material for pharmaceuticals.
The immunopotentiating action through the iNKT cell activating action of the sphingoglycolipid purified fraction, which is a rice bran extract component provided by the present invention, re-recognizes the importance of rice as a staple food by Japanese people, and many of them are agricultural In terms of finding new ways to use rice bran that has been treated as industrial waste, the contribution is significant.

Claims (10)

  An iNKT cell (invariant natural killer T cell) activator comprising rice bran, rice germ and / or glycosphingolipid derived therefrom.   The glycosphingolipid is composed of a purified glycosphingolipid fraction obtained by fractionating a crude glycosphingolipid-containing fraction obtained by extraction of rice bran, rice germ and / or materials derived therefrom with an organic solvent by chromatography and / or recrystallization. The iNKT cell activator according to claim 1.   The iNKT cell activator according to claim 2, wherein the organic solvent extraction is alcohol, ketone, chlorinated hydrocarbon and / or mixed solvent thereof, and / or water and mixed solvent extraction thereof.   The iNKT cell activator according to claim 2, wherein the chromatography is column chromatography.   The iNKT cell activator according to claim 2, wherein the chromatography is liquid chromatography.   The iNKT cell activator according to claim 2, wherein the recrystallization is alcohol, ketone and a mixed solvent thereof, and / or water and a mixed solvent thereof.   The iNKT cell activator according to claim 1, wherein the glycosphingolipid is β-glucosylceramide.   A medicament comprising the iNKT cell activator according to any one of claims 1 to 7.   A diagnostic agent comprising the iNKT cell activator according to any one of claims 1 to 7.   A functional food or drink comprising the iNKT cell activator according to any one of claims 1 to 7.

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