WO2005027937A1

WO2005027937A1 - Glycolipid-containing composition, use thereof and process for producing the same

Info

Publication number: WO2005027937A1
Application number: PCT/JP2004/013432
Authority: WO
Inventors: Yoshiyuki Mizushina; Takahiko Hada; Hiromi Yoshida
Original assignee: Yoshiyuki Mizushina; Takahiko Hada; Hiromi Yoshida
Priority date: 2003-09-22
Filing date: 2004-09-15
Publication date: 2005-03-31
Also published as: JP4744297B2; JPWO2005027937A1

Abstract

A glycolipid-containing composition which can be purified by a convenient method from, for example, spinach (Spinacia oleracea L.) and contains three glycolipids, i.e., monogalactosyl diacyl glyerol, digalactosyl diacyl glycerol and sulphoquinovosyl diacyl glycerol at a high ratio. This glycolipid-containing composition has useful physiological activities such as a DNA synthase inhibitory activity, a cancer cell growth inhibitory activity and an antitumor activity. When treated with a lipase, its shows further enhanced physiological activities and, therefore, becomes useful in, for example, functional foods having an anticancer effect or a cancer preventing effect.

Description

Specification

Glycolipid-containing composition, its use and production method thereof

Technical field

The present invention relates to a useful glycolipid-containing composition having a useful physiological activity, which can be used for medicines and foods (especially functional foods), its use, and its production method. Background art

[0002] 13 types of eukaryotic DNA synthases (DNA polymerases) have been known so far: α, β, _Ύ , δ, ε, ζ, η, θ, _Κ _,, ju, and σ. Have been. These DNA synthases are involved in cell growth, division, division, etc. α type is DN Α replication, | 8 type is repair and recombination, δ and ε types are replication and repair. It is known to have different functions depending on the type, such as carrying both.

[0003] Since DNA synthase is involved in cell growth and the like, a DNA synthase inhibitor that inhibits its enzymatic activity, for example, exhibits a cancer cell growth inhibitory effect on cancer, May have an inhibitory effect on HIV-derived reverse transcriptase, or an immunosuppressive effect on the production of specific antibodies against antigens in immunocompetent cells. Further, it is considered that the activity of the DNA synthase is inhibited to affect the cell cycle and to induce cell death (apoptosis). For this reason, DNA synthase inhibitors are expected to be used as anticancer agents (anticancer agents), AIDS therapeutic agents, antiviral agents, immunosuppressants, apoptosis inducers, etc. The development of pharmaceuticals that are effective in the prevention and treatment of various diseases such as viral diseases such as cancer and AIDS, and immune diseases, and the development of functional foods having similar effects are expected.

[0004] For example, it has been reported that glycolipids derived from red algae having DNA synthase inhibitory activity are useful as anticancer agents, HIV-derived reverse transcriptase inhibitors, and immunosuppressants (see Patent Document 1 below). ). Currently, dideoxy TTP (ddTTP), Ν-methylmaleimide, butylphenyl-dGTP, and the like are known as DNA synthase inhibitors (see Non-patent Document 1 below). In addition, sulfoquinovosylacylglycerol, which is a plant-derived glycolipid, has also been found to have a DNA synthetase inhibitory effect (see Patent Document 2 below). [0005] Furthermore, the antitumor effect of the glycolipid sulfoquinovosyl monoacylglycerol (SQMG) found from ゥ 2, monogalactosyldiasylglycerol (MGDG) purified from spinach (Spinacia oleracea L.), It has also been reported that various glycolipids such as digalatatosyldiacylglycerol (DGDG) and sulfoquinovosyldiacylglycerol (SQDG) inhibit DNA synthase (see Non-Patent Documents 2 and 3 below). 3).

[0006] Patent Document 1: Japanese Patent Application Laid-Open No. H11-106395

Patent Document 2: JP-A-2000-143516

Non-Patent Document 1: Annual Review of Biochemistry, 1991, 60, 513-552

Non-Patent Document 2: Jpn.J. Cancer Res., 2002, 93, pp. 85-92.

Non-Patent Document 3: Biochemical Pharmacology, 2003, 65, pp. 259-267

Disclosure of the invention

Problems to be solved by the invention

[0007] Recently, functional foods have been attracting attention as having preventive and ameliorating effects on various diseases such as lifestyle-related diseases. Functional foods contain natural ingredients such as AHCC (Active Hexose-Correlated Compound), shark cartilage, and agaritas.The physiological activity of these natural ingredients enhances human immunity and natural healing power, and prevents and improves disease. It is a plan. Therefore, it is conceivable to apply the above-mentioned glycolipids derived from natural products having DNA synthase inhibitory activity not only to pharmaceuticals but also to such functional foods. Since the glycolipids are purified independently, the purification is relatively complicated and time-consuming, and the yield is low, making them unsuitable for use in functional foods.

[0008] In view of the above circumstances, the present invention provides a glycolipid-containing composition that can be efficiently obtained from a natural product by a simple operation and that contains a glycolipid having useful physiological activity in high yield and high purity. And to provide its use and method of manufacture. Means for Solving the Problems

[0009] The present inventor has conducted intensive studies in view of the above-mentioned problems, and as a result, (1) a glycolipid containing three types of glycolipids described below in high yield and high purity by devising the production conditions. composition (2) The resulting glycolipid-containing composition has DNA synthase inhibitory activity, cancer cell growth inhibitory activity, and antitumor activity in vivo. (3) Glycolipid-containing composition containing at least two monoacyldarcerol-type glycolipids by subjecting the glycolipid-containing composition to lipase treatment Thus, the composition was found to have more potent DNA synthase inhibitory activity, cancer cell growth inhibitory activity, antitumor activity, and the like, thereby completing the present invention.

That is, the present invention includes the following industrially useful inventions A) to L).

A) A seaweed or terrestrial plant power is also purified, and a glycolipid-containing composition containing at least monogalactosyl sialyl glyceryl glycerol, digalatatosyl diacyl glycerol, and sulfoquinovosyl diacyl glycerol as glycolipids (hereinafter, particularly, `` It may be referred to as "the first glycolipid-containing composition").

B) A glycolipid-containing composition obtained by subjecting the first glycolipid-containing composition to lipase treatment and containing at least monogalactosyl monoacylglycerol and sulfoquinovosylmonoacylglycerol as glycolipids (hereinafter, particularly, It may be referred to as “the second glycolipid-containing composition”).

C) A DNA synthase inhibitor comprising the first or second glycolipid-containing composition as an active ingredient.

D) An anticancer agent comprising the first or second glycolipid-containing composition as an active ingredient.

E) A pharmaceutical composition comprising the first or second glycolipid-containing composition as an active ingredient.

F) An edible composition comprising the first or second glycolipid-containing composition.

G) A method for producing a glycolipid-containing composition containing seagrass or a terrestrial plant as a raw material and containing at least glycolipid sildiacylglycerol, digalatatosyldiacylglycerol, and sulfoquinovosyldiacylglycerol as glycolipids.

H) A glycolipid-containing composition containing at least monogalactosyl monoacylglycerol and sulfoquinovosyl monoacylglycerol as glycolipids by subjecting the glycolipid-containing composition produced by the method described in G) to lipase treatment. How to make things.

I) In the method described in G) above, the plant extract (sea Meaning of extracts from algae or terrestrial plants. same as below. A) purifying the glycolipid fraction from the above).

J) In the method described in I) above, use an alcohol such as ethanol or an organic solvent such as acetone to elute the glycolipid, and elute the water-soluble substance with a 50% -75% water-containing organic solvent. — A method including the step of eluting glycolipids with 100% aqueous organic solvent or organic solvent.

K) The method according to the above G), comprising a step of washing the plant with warm water at 40 ° C to 80 ° C to remove water-soluble components before obtaining the plant extract.

L) A method using green-yellow vegetables such as spinach (Spinacia) as a raw material according to the method described in G) above.

The invention's effect

[0011] Both the first and second glycolipid-containing compositions of the present invention have useful physiological activities such as DNA synthase inhibitory activity, cancer cell growth inhibitory activity, and antitumor activity. , An anticancer agent and other pharmaceutical compositions and edible compositions (food or food additives). Moreover, the glycolipid-containing composition of the present invention can be efficiently produced by simple operation of seaweed or terrestrial plant, and can be produced in large quantities and at low cost because of its high yield. It can be suitably used as, for example, a functional food having an anticancer effect and a cancer preventive effect.

Brief Description of Drawings

FIG. 1 (a)-(e) shows the chemical structures of various glycolipids contained in the glycolipid-containing composition of the present invention.

FIG. 2 is a diagram illustrating a method for producing the first glycolipid-containing composition of the present invention.

FIG. 3 is a view showing a result of analyzing components contained in the first glycolipid-containing composition of the present invention by thin-layer chromatography.

FIG. 4 is a graph showing the results of examining the DNA synthetase a inhibitory activity of the first glycolipid-containing composition of the present invention.

FIG. 5 is a graph showing the results of examining the cancer cell growth inhibitory activity of the first glycolipid-containing composition of the present invention. FIG. 6 is a view showing a result of analyzing components contained in the second glycolipid-containing composition of the present invention by thin-layer chromatography.

FIG. 7 is a graph showing the results of examining DNA synthase inhibitory activities of the first and second glycolipid-containing compositions of the present invention.

FIG. 8 is a graph showing the results of examining the cancer cell growth inhibitory activity of the first and second glycolipid-containing compositions of the present invention.

FIG. 9 is a graph showing the results of examining the in vivo antitumor activity of the first and second glycolipid-containing compositions of the present invention.

[FIG. 10] FIG. 10 is a diagram showing the results of examining in vivo antitumor activity in the same manner as in FIG. 9, wherein (a) shows the results obtained when physiological saline was administered, and (b) shows the results obtained when the second glycolipid-containing composition was used. This is the result of administration.

FIG. 11 is a graph showing the results of investigating the in vivo antitumor activity of the glycolipid-containing composition of the present invention in nude mice (balbZc, —Z—) transplanted with human child cancer cells (HeLa).

FIG. 12 is a graph showing the results of investigating the in vivo antitumor activity of an ICR mouse transplanted with a mouse-derived sarcoma cell (S-180) when the glycolipid-containing composition of the present invention was orally administered. .

FIG. 13 is a diagram illustrating a method for producing a second glycolipid-containing composition by treating the first glycolipid-containing composition with lipase.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, an embodiment of the present invention will be described with reference to the drawings.

(1) Glycolipid-containing composition of the present invention and method for producing the same

As described above, the first glycolipid-containing composition of the present invention is purified from seaweed or a terrestrial plant, and contains at least glycolipid monogalactosyldiasylglycerol, digalatatosyldiasylglycerol, and sulfoquinovosyldiacil. It contains glycerol (hereinafter, these glycolipids are abbreviated as “MGDG”, “DGDG”, and “SQDG”, respectively). Further, the second glycolipid-containing composition of the present invention is obtained by subjecting the first glycolipid-containing composition of the present invention to lipase treatment, and at least monogalactosyl monoacyl as a glycolipid. It contains glycerol and sulfoquinovosylmonoacylglycerol (hereinafter, these glycolipids are abbreviated as “MGMG” and rsQMGj, respectively).

FIGS. 1 (a) to 1 (e) show the chemical structures of the above MGDG, DGDG, SQDG, MGMG, and SQMG, respectively. In the figure, R—R are fatty acids independent of each other, preferably

1 8

It is a saturated fatty acid having 14 to 22 carbon atoms or a mono- or poly-unsaturated fatty acid. Examples of the saturated fatty acid include myristic acid, palmitic acid, and stearic acid, and examples of the unsaturated fatty acid include palmitooleic acid, oleic acid, linoleic acid, linolenic acid (ω3), dihomoganmarinolenic acid, and octadedecaic acid. Examples thereof include tetraenoic acid, arachidonic acid, eicosapentaenoic acid, docosapentaenoic acid, and docosahexaenoic acid. The types of the fatty acids constituting the first and second positions of the glycerol skeleton in each glycolipid are not particularly limited.In the case of diacyl darcerol type glycolipids, even if both fatty acids are the same. , May be different.

Further, each glycolipid may be in the form of a pharmaceutically acceptable salt. Examples of such salts include halogenated hydrochlorides such as hydrofluorides and hydrochlorides, inorganic salts such as sulfates and nitrates, alkali metal salts such as sodium and potassium salts, sulfonates, and organic salts. Acid salts and amino acid salts, preferably, hydrochloride, sulfate, nitrate, sodium salt and potassium salt.

[0015] The first glycolipid-containing composition of the present invention can be prepared, for example, by the following method.

Spinacia) Power Extraction * Can be purified. First, a commercially available spinach (Spinacia oleracea L.) was used as a raw material. As shown in Fig. 2, 100 g of the dried product was shredded, and hot water at 60 ° C was used to remove as much water-soluble components as possible. Wash twice with 1L. Next, after filtering using filter paper, water is removed, and the obtained solid (residue) is heated with 1 L of ethanol, and reflux extraction is performed twice at 60 ° C with stirring. The extract is filtered and then concentrated under reduced pressure to obtain an oily extract of spinach. In fact, this method yielded 20.5 g of oily extract from 100 g of dried spinach.

Next, the obtained extract is dissolved in a 70% ethanol solution (a solution having a volume ratio of ethanol and water of 70:30; the same applies hereinafter), and the glycolipid fraction is purified by reverse phase chromatography. Then, the first glycolipid-containing composition of the present invention is obtained. In the method performed by the inventor, the above extraction After dissolving the substance in a 70% ethanol solution, this was injected into 500 g of a resin for hydrophobic chromatography (Diaion HP-20, manufactured by Mitsubishi Chemical Corporation), and then unadsorbed substances were washed with 70% ethanol and eluted. Fraction 1 (water-soluble fraction, 13.0 g), fraction eluted with 95% ethanol Π (glycolipid fraction, ie, the glycolipid-containing composition of the present invention, 6.5 g), eluted with black-mouthed form Fraction ΠΙ (a pigment fraction containing chlorophyll (chlorophyll), 1. Og).

[0017] Analysis of the above-mentioned fraction II, ie, the components contained in the glycolipid-containing composition of the present invention, by thin-layer chromatography confirmed that it contained three glycolipids, MGDG, DGDG and SQDG. (See Figure 3). As a result of analysis, 250 mg of the glycolipid fraction (Fraction II) contained 84.7 mg (33.9%) of MGDG, 35.0 mg (14.0%) of DGDG, and 92.3 mg (36.9%) of SQDG. Was. Thus, in the first glycolipid-containing composition of the present invention, the content of the three glycolipids MGDG, DGDG and SQDG is preferably at least 70% by weight, more preferably at least 75%, still more preferably at least 75%. Is more than 80%.

[0018] Furthermore, when the above-mentioned glycolipid fraction (Fraction II) was examined for its DNA synthase inhibitory activity and human cancer cell growth inhibitory activity, the glycolipid fraction inhibited DNA synthase α, and It inhibited the growth of strain NUGC-3 cells and also exhibited antitumor activity in vivo (see FIGS. 4, 5 and 9 and 12). The details of these experiments will be described in Examples below.

As described above, the glycolipid-containing composition of the present invention can be extracted and purified from spinach by the above-described method, but various modifications other than the above-described production method are possible. For example, although spinach (Spinacia oleracea) was used as a raw material in the above purification method of Fraction II, other plants or seaweeds may be used as a raw material. Preferably, other green and yellow vegetables (komatsuna, thalesone, parsley, broccoli, peppers, etc.) and terrestrial products such as barley leaves, cyanobacteria (Anabaena vanaDilis, Anacystis nidulans, Spirulina platensis: spinorelina, etc.), and filamentous algae (Porphyra) yezoensis: Susabinori, Gelidium amansn: Maxa, Gigartina tenella: Suginori, Gracilaria verrucosa: Ogonori, brown algae (Undaria pinnatinda: Wakame), Hizikia! sp.: A kind of Chlamydomonas, Enteromorpha sp.: A kind of aosa, Chlorella sp.: Chlorella sp. Can be mentioned.

[0020] One or more of these raw materials are shredded or pulverized as necessary, and an organic solvent for extracting lipid components such as hexane, black form, acetone, methanol, or ethanol is used. To extract and remove the extractive solvent to obtain total lipids. Subsequently, the concentrated fraction was collected by fractionation by column chromatography using silica gel, alumina, cefadettas, a reversed-phase adsorbent (such as octadecylsilylido silica gel), ion-exchange resin, and a synthetic adsorbent. Further, if necessary, the composition can be concentrated and purified to prepare the glycolipid-containing composition of the present invention, which is the target substance. Furthermore, the second glycolipid-containing composition of the present invention containing a monoacylglycerol-type glycolipid can be prepared by subjecting the obtained glycolipid-containing composition to lipase treatment as described below. .

[0021] More specifically, two preferable methods for producing the glycolipid-containing composition of the present invention from a plant will be described. In the first method, first, a dried product of a plant is shredded. Wash with warm water, preferably 40 ° C-80 ° C (more preferably 50 ° C-70 ° C). Thereafter, water is removed by filtration, and a 50% or more ethanol solution or ethanol is added to the obtained solid (residue), and the mixture is stirred at 40 ° C to 80 ° C (more preferably at 50 ° C to 70 ° C). Perform reflux extraction in C). After the extract is filtered, the extract is concentrated under reduced pressure, and the obtained extract is dissolved in a 50% -75% ethanol solution and injected into a hydrophobic chromatography resin. Elution is performed stepwise at a water-ethanol mixing ratio. First, a 50% -75% ethanol solution elutes water-soluble substances that do not adsorb to the fat, followed by 85% or more ethanol solution (or an organic solvent such as acetone) elutes lipid-soluble substances such as glycolipids. Is done. Furthermore, glycolipid fractions may be purified by removing fat-soluble dyes and fatty acids, but it is possible to efficiently fractionate only glycolipids by eluting with 85% -100% ethanol solution. The aforementioned method of purifying Fraction II performed by the present inventors is also basically the first method, and is preferred as a method for preparing the glycolipid-containing composition of the present invention simply, quickly, with high purity and high yield. Well, the way.

[0022] In the second method, first, a dried product of a plant is cut into small pieces, water-containing acetone or acetone is added, and reflux extraction is performed at 30 to 50 ° C with stirring. The extract is filtered, concentrated under reduced pressure, and the obtained extract is dissolved in a water-containing acetone solution, and ion-exchange resin (SK1B ion exchange) is used. (For example, manufactured by Mitsubishi Idani Gakusha). By performing the elution stepwise at a mixing ratio of water-acetone, the glycolipid-containing fraction can be obtained, for example, by elution with a 95% or more acetone solution.

[0023] The above-described purification method of Fraction II performed by the present inventor is as follows: (1) Before obtaining a crude extract (oil-like extract), the water-soluble component is extracted as much as possible by washing with warm water, that is, washing with warm water. The removal of the glycolipid fraction facilitated purification of the glycolipid-containing composition, (2) the use of hydrophobic chromatography, and (3) the ratio of water / ethanol when the glycolipid fraction was eluted with an ethanol solution. Strictly studied, is characterized. For (1) above, plants (spinach) may be washed twice with 1 L of hot water at 60 ° C, and washed with hot water at a power of 40 ° C to 80 ° C, from which water-soluble components have been removed. Further, the amount of hot water, the number of washings, and the like may be appropriately set according to the amount of raw materials and the like. Regarding the above (2), hydrophobic (reverse phase) chromatography may be carried out using a force such as Diaion HP-20 manufactured by Mitsubishi Dani Gakusha Co., Ltd., or another resin. For (3) above, the ability to elute a water-soluble fraction (Fraction I) with a 70% ethanol solution and then elute a glycolipid fraction (Fraction II) with a 95% ethanol solution. After eluting the water-soluble substance with 50% to 75% (more preferably 65% to 75%) of a water-containing organic solvent using an alcohol such as alcohol or acetone, 85% to 100% (more preferably The glycolipid may be eluted with a water-containing organic solvent or an organic solvent (92% to 98%).

[0024] As described below, Fraction II obtained by the above method has activities such as DNA synthase inhibitory activity and cancer cell growth inhibitory activity, while a simple ethanol extract (the above-described oily state) is used. Extract), and no such activity was observed in the water-soluble fraction (fraction I) and the pigment fraction (fraction III). From the results of these experiments, it is assumed that the activity-interfering substance (masking substance) may be present in the water-soluble fraction or the pigment fraction.If so, the glycolipid fraction was purified by the method described above, Removing the interfering substance is a very effective method. Further, producing the glycolipid-containing composition of the present invention by the above method is easier than (1) purifying each glycolipid, and (2) yielding more than purifying each glycolipid. (3) It is easier to use as a functional food than purified glycolipids.

[0025] In the first glycolipid-containing composition of the present invention, each of MGDG, DGDG and SQDG The ratio of each of them is not particularly limited. The content ratio of these three glycolipids differs depending on the plant 'seaweed' used as a raw material. For example, in the case of spinach, the content ratio of these glycolipids is usually MGDG: DGDG: SQDG = about 1: 2.15: 1.75 (about 20:43 : 35). In the case of other vegetables and the like, the content ratio of these glycolipids may be very different. According to the results of experiments performed by the present inventors, the higher the ratio of SQDG and the higher the glycolipid fraction, the stronger the activity of inhibiting DNA synthase, so the higher the SQDG content, the better.

[0026] The type of fatty acid chain of each glycolipid contained in the glycolipid-containing composition of the present invention is not particularly limited, but it is preferable that the fatty acid chain is relatively long (for example, having 16 or more carbon atoms). Since the DNA synthetase inhibitory activity is high when the number of double bonds in the unsaturated fatty acid chain is one or two, it is preferable that both are different.

[0027] As described above, in the first glycolipid-containing composition of the present invention, the content of the three glycolipids MGDG, DGDG and SQDG is preferably 70% or more by weight, more preferably 75% or more. It is more preferably 80% or more, and it is preferable that components other than glycolipids are not contained as much as possible. However, since the flavonoid conjugate having antioxidative activity (lipid-soluble component such as power techin porifenol) has the property of preventing oxidation of the double bond of fatty acid, the glycolipid-containing composition of the present invention May contain a substance such as the same compound.

[0028] Furthermore, the first glycolipid-containing composition of the present invention obtained by the above-described production method is subjected to a revase treatment, whereby the second glycolipid of the present invention containing a monoacylglycerol-type glycolipid is obtained. A glycolipid-containing composition can be produced.

[0029] As shown in Fig. 13, the method performed by the present inventor was to react the above-mentioned glycolipid fraction (fraction II) lOmg / ml and porcine spleen lipase lOmgZml with a reaction solution (0.2M Tris-HCl (pH 7.6) ), 0.25 M CaCl 2) at 37 ° C for 20 minutes with shaking reaction to hydrolyze glycolipids with lipase,

2

The reaction is then stopped with 6N HC1. Next, by partitioning with n-butanol (n-BuOH) and water, the glycolipid moves to the n-butanol layer and the lipase moves to the aqueous layer, so that the second lipid of the present invention containing the lipase-degraded glycolipid is contained. Glycolipid-containing composition can be prepared

[0030] As a result of analysis by thin-layer chromatography, the glycolipid-containing composition after the lipase reaction contained two glycolipids, MGMG and SQMG. The glycolipid MGDG'SQDG in II was decomposed into MGMG'SQMG by a lipase hydrolysis reaction. On the other hand, DGDG was not hydrolyzed by lipase and was also detected in the glycolipid-containing composition after the lipase reaction (see FIG. 6).

[0031] As described above, the second glycolipid-containing composition of the present invention obtained by treating the first glycolipid-containing composition of the present invention with lipase was examined for its DNA synthase inhibitory activity and the like. Higher activity was observed in all of DNA synthase inhibitory activity, cancer cell growth inhibitory activity, and antitumor activity than the first glycolipid-containing composition (see FIGS. 7 to 9). Therefore, it can be said that the second glycolipid-containing composition of the present invention is more effective for various uses such as a DNA synthase inhibitor, an anticancer agent (an anticancer agent), and a functional food.

The lipase treatment for obtaining the first glycolipid-containing composition of the present invention and the second glycolipid-containing composition is not limited to the method shown in FIG. The second glycolipid-containing composition may be produced by the lipase treatment.

When the first and second glycolipid-containing compositions of the present invention are industrially produced, the specific method of each step is appropriately changed in order to make the above-described method more suitable for industrial production. You may get. For example, in the process of purifying the glycolipid fraction by hydrophobic chromatography, besides the column method (a method of packing chromatographic resin in a cylindrical column and flowing the composition solution), the Notchi method (the method of It is considered that the method of adding chromatographic resin inside and mixing vigorously) is advantageous for industrialization. As for the lipase treatment, in addition to a method in which an enzyme (lipase) solution is added to a composition solution and reacted while stirring, a so-called “immobilized enzyme enzyme” is used in which the composition is allowed to flow through a column on which lipase is immobilized. Is considered to be advantageous for mass production.

(2) Use of the glycolipid-containing composition of the present invention

Since the above-mentioned first and second glycolipid-containing compositions of the present invention have useful physiological activities such as DNA synthase inhibitory activity, cancer cell growth inhibitory activity, and antitumor activity, DNA synthases It can be used as an inhibitor or an anticancer agent (an anticancer agent), and can be suitably used particularly for functional foods and the like.

[0035] Further, the first and second glycolipid-containing compositions of the present invention have a DNA synthase inhibitory activity. As a result, it can be used for medicines (pharmaceutical compositions) other than anticancer drugs and as edible compositions (foods or food additives) other than functional foods. The preventive or therapeutic effect and the immunosuppressive effect at the time of organ transplantation can be used as the aim, and it is particularly useful as the AIDS therapeutic agent, antiviral agent, immunosuppressant, apoptosis inducer, and the like. As described above, the glycolipid-containing composition of the present invention can be used as a means for preventing or treating cancer, AIDS and other viral infections, immune diseases, and the like.

As an example, it has been demonstrated that a commercially available clinical drug such as azidothymidine, a DNA synthase inhibitor, can also have a therapeutic effect on AIDS (edited by Japan Pharmaceutical Information Center, Pharmaceutical Co., Ltd.) Published by Jikhosha, 1996 Pharmaceutical Drugs, Japan Pharmaceutical Collection, p. 599).

Next, a pharmaceutical composition and an edible composition containing the glycolipid-containing composition of the present invention will be described. The glycolipid-containing composition of the present invention can be further purified, if necessary, and then used as it is or as a pharmaceutical composition together with a conventional pharmaceutical preparation carrier, and can be administered to animals and humans. The dosage form of the pharmaceutical composition is not particularly limited and may be appropriately selected as needed. Examples thereof include oral preparations such as tablets, capsules, granules, fine granules and powders, injections, Parenteral preparations such as suppositories are included. In general, it is appropriate for adults to take 20 mg or more and 600 mg or less per day as glycolipid in several divided doses.

[0038] In the present invention, oral preparations as tablets, capsules, granules, fine granules and powders include, for example, starch, lactose, sucrose, mannitol, carboxymethylcellulose, corn starch, inorganic salts and the like. And is manufactured according to a conventional method. The amount of glycolipid in these preparations is not particularly limited and can be appropriately designed. In this type of preparation, in addition to the glycolipid-containing composition of the present invention, a binder, a disintegrant, a surfactant, a lubricant, a fluidity promoter, a flavoring agent, a coloring agent, a flavor, and the like are appropriately used. be able to.

[0039] Examples of the binder include starch, dextrin, gum arabic powder, gelatin, hydroxypropyl pill starch, methylcellulose sodium, hydroxypropylcellulose, crystalline cellulose, ethylcellulose, polybutylpyrrolidone, macrogol and the like. Collapse Examples of the disintegrant include starch, hydroxypropyl starch, sodium carboxymethylcellulose, calcium carboxymethylcellulose, carboxymethylcellulose, low-substituted hydroxypropylcellulose and the like. Examples of the surfactant include sodium lauryl sulfate, soybean lecithin, sucrose fatty acid ester, polyoxyethylene sorbitan fatty acid ester and the like. Examples of the lubricant include talc, waxes, hydrogenated vegetable oil, sucrose fatty acid ester, magnesium stearate, calcium stearate, aluminum stearate, polyethylene glycol and the like. Examples of the fluidity promoter include light anhydrous silicic acid, dried hydroxide aluminum gel, synthetic aluminum silicate, magnesium silicate and the like. The glycolipid-containing composition can also be administered as a suspension, emulsion, syrup, and elixir. These various forms may contain a flavoring agent and a coloring agent.

[0040] In order to achieve the desired effect of the present invention as a parenteral agent, a force that varies depending on the age, weight, and degree of the disease of the patient is usually 1 to 60 mg per day as a glycolipid in an adult by intravenous injection. Intravenous infusion, subcutaneous injection, and intramuscular injection are appropriate. This parenteral preparation is manufactured according to a conventional method, and is generally used as a diluent, such as distilled water for injection, physiological saline, aqueous glucose solution, vegetable oil for injection, sesame oil, laccase oil, soybean oil, corn oil, propylene glycol, etc. Can be used. Further, if necessary, a bactericide, a preservative, and a stabilizer may be added. In addition, from the viewpoint of stability, this parenteral preparation can be frozen after filling into a vial or the like, and water can be removed by a usual freeze-drying treatment. Further, if necessary, an isotonic agent, a stabilizer, a preservative, and a soothing agent may be added. The amount of the glycolipid-containing composition in these preparations is not particularly limited and can be set arbitrarily. Examples of other parenteral preparations include liquid preparations for external use, ointments such as ointments, suppositories for rectal administration, and the like, which are also produced according to a conventional method.

[0041] Another preferred use of the glycolipid-containing composition of the present invention is an edible composition. That is, the glycolipid-containing composition of the present invention is further purified as necessary, and then is directly used as a liquid, gel or solid food, such as juice, soft drink, tea, soup, soy milk, salad oil, Add to dressing, yogurt, jelly, pudding, sprinkle, infant formula, cake mix, powdered or liquid dairy products, bread, cookies, etc. Processed into pellets, tablets, granules, etc. together with excipients such as sugar, lactose, starch, etc. and flavors, pigments, etc., or coated with gelatin etc. to form capsules and processed into health foods, dietary supplements, and functional foods. Available as etc.

[0042] The amount of the glycolipid-containing composition of the present invention in these foods or edible compositions cannot be uniformly defined depending on the type and condition of the food or composition, but is preferably about 0.01 to 50%. %, More preferably 0.1-30% by weight. If the amount is less than 0.01% by weight, the desired effect of ingestion is small. If it exceeds 50% by weight, the flavor may be impaired or the food may not be prepared depending on the type of food.

Example

Hereinafter, embodiments of the present invention will be described with reference to the drawings, but the present invention is not limited to these embodiments.

[Example 1: Purification of the first glycolipid-containing composition (glycolipid fraction) of the present invention]

In the present example, the first glycolipid-containing composition of the present invention was purified from spinach (Spinacia oleracea) as a raw material by the method shown in FIG. First, 100 g of dried spinach was shredded and washed twice with 1 L of hot water at 60 ° C to remove water-soluble components as much as possible. Next, after filtration using a filter paper, water was removed, 1 L of ethanol was added to the obtained solid (residue), and reflux extraction was performed twice at 60 ° C with stirring. The extract was filtered and concentrated under reduced pressure to obtain 20.5 g of an oily extract of spinach.

Next, the obtained extract was dissolved in a 70% aqueous ethanol solution and injected into 500 g of resin for hydrophobic chromatography (Diaion HP-20, manufactured by Mitsubishi Chemical Corporation). Thereafter, unadsorbed substances were washed with 70% ethanol, and fraction 1 eluted (water-soluble fraction, 13.0 g) and fraction II eluted with 95% ethanol (glycolipid fraction, ie, the sugar of the present invention) Lipid-containing composition, 6.5 g), and fraction III (dye fraction, 1.0 Og) eluted with black-mouthed form.

FIG. 3 is a diagram showing the results of analyzing the components contained in the above fractions I-III by thin-layer chromatography. In thin-layer chromatography, 2 g of a sample was added to the starting point of a silica gel plate, and the mixture was developed with black-mouth form: methanol = 3: 1. In the figure, lanes 1-4 are the detection results of ethanol extract (the above oily extract; the same applies hereinafter), fraction III, fraction I, and fraction II, respectively. As shown in the figure, The fraction in lane 4 was found to contain three glycolipids, MGDG, DGDG and SQDG. On the other hand, none of these glycolipids was detected in fractions I and III.

Next, MG DG, DGDG and SQDG were isolated and purified from the glycolipid fraction Fraction II using silica gel column chromatography. As a result, it was found that 250 mg of the glycolipid fraction contained 84.7 mg (33.9%) of MGDG, 35.0 mg (14.0%) of DGDG, and 92.3 mg (36.9%) of SQDG. Furthermore, the purified MGDG, DGDG, and SQDG were subjected to structure confirmation and purity testing using ¹ H-NMR and ¹³ C-NMR, and as a result, the chemical structure was confirmed for both MG DG, DGDG, and SQDG. In each case, it was over 95%.

Example 2 DNA Synthase Inhibitory Activity of the Glycolipid Fraction

The DNA synthase inhibitory activity of the glycolipid fraction (fraction Π) obtained by the above method, ie, the first glycolipid-containing composition of the present invention, was examined. Mammalian DNA synthases oc and β were used as DNA synthases. In more detail, DNA synthase oc is a sample obtained by purifying bovine thymus extract by antibody column chromatography, and DNA synthase | 8 is a recombinant E. coli transfected with rat DΝΑ synthase β gene. After culturing, a crushed and purified preparation was used.

[0049] In order to measure the inhibitory effect on DNA synthase a and β, a general DNA synthase reaction system (edited by The Biochemical Society of Japan, Lectures on Experimental Chemistry 2, Nucleic Acid IV, Tokyo Kagaku Dojin, p. 63-1 66 page). That is, a DNA synthesis reaction is performed in a system containing [ ³ [] -ΤΤΡ labeled with a radioisotope, and the radioactivity is used as an indicator of the amount of the product (synthetic DNA chain). The inhibition rate was calculated based on (a) the amount of synthetic DNA in the control, and (b) the amount of synthetic DNA in the presence of the spinach fraction (or ethanol extract) to be investigated.

(a-b) / a X 100 = inhibition rate (%)

Was evaluated. The results obtained are shown in FIG. The figure shows the results of inhibition of DNA synthase oc.In the figure, triangles indicate the results of inhibition in the presence of ethanol extract, and diamonds, circles, and squares indicate the presence of fractions I-III, respectively. The inhibition results below. As shown in the figure, fraction II showed strong DNA synthase α inhibitory activity, and 50% of fraction II The inhibitory concentration (IC) was approximately gZml. On the other hand, ethanol extract, fraction I and

50

And III hardly inhibited DNA synthase α.

Example 3 Human Cancer Cell Growth Inhibitory Activity of the Glycolipid Fraction

Next, it was examined whether each of the spinach fractions and the ethanol extract had an inhibitory effect on cancer cell growth. In the experiment, NUGC-3 cells, a human gastric cancer cell line, were incubated with various concentrations of spinach grass fractions (or ethanol extracts), and after 48 hours, the cancer cells in each case were incubated. The survival rate was determined by the usual PI.

FIG. 5 shows the results. In the figure, triangles indicate the results of inhibition of growth in the presence of ethanol extract, and diamonds, circles, and squares indicate the results of inhibition of growth in the presence of fractions I-III. As shown in the figure, Fraction II showed a strong effect on human stomach cancer cells and showed growth inhibitory activity. The ethanol extract and Fractions I and III showed no growth suppressing activity. This result was in good agreement with the experimental results of the DNA synthase oc inhibitory activity.

Example 4: Purification of Second Glycolipid-Containing Composition by Lipase Treatment of the Glycolipid Fraction The above-mentioned glycolipid fraction (fraction II) was treated with lipase by the following method, Was purified.

As shown in FIG. 13, lOmgZml of the glycolipid fraction (Fraction II) and 10 mgZml of porcine spleen lipase were shaken in a reaction solution (0.2 M Tris-HCl (pH 7.6), 0.25 M CaCl 2) at 37 ° C for 20 minutes. When

2

Glycolipids were hydrolyzed with lipase to produce diacylglycerol-type mosquitoes and monoacylglycerol-type mosquitoes, and then the reaction was stopped with 6N HC1

[0053] Next, by partitioning with n-butanol (n-BuOH) and water, the glycolipid moves to the n-butanol layer and the lipase moves to the aqueous layer. 2) can be purified.

FIG. 6 shows the components contained in the glycolipid fraction (fraction Π) before the lipase reaction (first glycolipid-containing composition) and after the lipase reaction (second glycolipid-containing composition), respectively. FIG. 4 is a view showing the result of analyzing the same by thin-layer chromatography as in FIG. In the figure, lane 1 shows the detection results before the lipase reaction, and lane 2 shows the detection results after the lipase reaction. As shown in the figure, The second glycolipid-containing composition contains two glycolipids, MGMG and SQMG, and the glycolipid MGDG ′ SQDG in the first glycolipid-containing composition is converted to MGMG and SQMG, respectively, by a lipase hydrolysis reaction. 'Disassembled into SQMG. On the other hand, DGDG was not hydrolyzed by lipase and was detected in the second glycolipid-containing composition.

Example 5 DNA Synthase Inhibitory Activity and Human Cancer Cell Growth Inhibitory Activity of Second Glycolipid-Containing Composition

The DNA synthesis of the glycolipid fraction before the lipase treatment (the first glycolipid-containing composition) and the glycolipid fraction after the lipase treatment (the second glycolipid-containing composition) were performed in the same manner as in the experiment of FIG. The synthase inhibitory activity was examined and both were compared. Figure 7 shows the results. In the figure, black circles indicate the glycolipid fraction before lipase treatment for DNA synthase oc, black squares indicate the glycolipid fraction after lipase treatment for DNA synthase ex, and white circles indicate DNA synthase β. The glycolipid fraction before the lipase treatment and the open squares indicate the results of inhibition of the DNA lipid synthesizing enzyme | 8 by the glycolipid fraction after the lipase treatment.

[0056] As shown in the figure, both the glycolipid fractions before and after the lipase treatment have a stronger DNA synthase inhibitory activity against DNA synthase oc than DNA synthase β. Was. In addition, the glycolipid fraction after the lipase treatment (that is, the second glycolipid-containing composition) is better than the glycolipid fraction before the lipase treatment (that is, the first glycolipid-containing composition). It strongly inhibited oc and β.

Next, by the same method as in the experiment of FIG. 5, the glycolipid fraction before the lipase treatment (the first glycolipid-containing composition) and the glycolipid fraction after the lipase treatment (the second glycolipid fraction) Containing composition) was examined for its inhibitory effect on human cancer cell proliferation, and both were compared. Fig. 8 shows the results. In the figure, solid circles indicate the results of the glycolipid fraction before lipase treatment, and solid squares indicate the results of the glycolipid fraction after lipase treatment. As shown in the figure, the glycolipid fraction after the lipase treatment (that is, the second glycolipid-containing composition) was better than the glycolipid fraction before the lipase treatment (that is, the first glycolipid-containing composition). ) More strongly inhibited and inhibited the growth of human gastric cancer cells (NUGC-3).

[0058] As described above, the lipase-treated second glycolipid-containing thread composition has a higher DNA synthase inhibitory activity and a higher level of human cancer cells than the first glycolipid-containing composition not treated with lipase. Proliferation inhibitory activity! Example 6 Antitumor Activity of First and Second Glycolipid-Containing Compositions of the Present Invention Further, the in vivo antitumor activity of the first and second glycolipid-containing compositions was investigated. . Syrian (Golden) hamsters were used as experimental animals, and melanoma (Mel anotic No. 179, Dl-179) pieces were subcutaneously transplanted into the hamsters, allowed to settle for one week, and then physiologically treated with glycolipid-containing compositions. After dissolving in saline, lOmgZkg was administered by subcutaneous injection every other day, and the tumor volume was measured. The results are shown in FIGS. 9 and 10.

[0060] In the graph of Fig. 9, white squares represent controls, and the results obtained when physiological saline was administered. The black triangles, black circles, and black diamonds represent the results before and after the ethanol extract and lipase treatment, respectively. FIG. 9 shows the results obtained when a glycolipid fraction (first glycolipid-containing composition) and a glycolipid fraction after lipase treatment (second glycolipid-containing composition) were administered. Similarly to the DNA synthetase inhibitory activity and the human cancer cell growth inhibitory activity, the anti-tumor activity of the second glycolipid-containing composition was stronger than that of the first glycolipid-containing composition. FIG. 10 (a) shows the results obtained when physiological saline was administered, and FIG. 10 (b) shows the results obtained when the second glycolipid-containing composition was dissolved and administered in physiological saline. In the nomster to which the glycolipid-containing composition No. 2 was administered, tumor hypertrophy was strongly suppressed.

[0061] Further, the in vivo antitumor activity of the glycolipid-containing composition of the present invention was investigated by an experiment in which a human-derived tumor was transplanted into a mouse. FIG. 11 shows the results of investigating the in vivo antitumor activity of the glycolipid-containing composition of the present invention in nude mice (balb, c, − / −) transplanted with human ovarian cancer cells (HeLa). In the experiment, the tumor volume reached 25 mm ³ on the 17th day after subcutaneous injection of human human ovarian cancer cells (HeLa) from nude mice (balbZc, —Z—). The fraction II (the first glycolipid-containing composition) or the fraction after the lipase treatment (the second glycolipid-containing composition) was dissolved in physiological saline and injected subcutaneously. . The glycolipid-containing composition of the present invention was dissolved in physiological saline at a ratio of 5 mgZml, and the glycolipid-containing composition of the present invention was administered to lOmgZkg mice each time. As a result, as shown in Fig. 11, compared with the control injected with physiological saline alone, the mice injected with fraction II of spinach and fraction II after lipase treatment showed suppression of tumor hypertrophy. . In addition, fraction II after lipase treatment had stronger antitumor activity than fraction II of spinach. From the above results, the glycolipid-containing composition of the present invention has an inhibitory activity on human-derived tumors as well as hamster-derived tumors, and is therefore useful for the development of health foods and the like.

[0062] Next, it was examined whether or not the glycolipid-containing composition of the present invention showed in vivo antitumor activity when administered orally. FIG. 12 shows the results of investigating the in vivo antitumor activity of the glycolipid-containing composition of the present invention in an ICR mouse transplanted with sarcoma cells (S-180) derived from a mouse. The experiment, after the mouse-derived sarcoma cells (S- 180) transplant Ri by the subcutaneous injection, where the tumor volume became 100mm ³ to 4 days, once a day, a fraction II (the first of sugar of spinach Lipid-containing composition) was dissolved in physiological saline and orally administered. The glycolipid-containing composition of the present invention was dissolved in physiological saline at a rate of 5 mgZml, and the glycolipid-containing composition of the present invention was administered to 70 mgZkg mice each time. As a result, as shown in FIG. 12, compared with the control fed only saline, mice with orally administered fraction II of spinach showed significant antitumor activity at day 6 and almost 30 days The tumor disappeared.

From the above results, the glycolipid-containing composition of the present invention is useful for the development of an anticancer functional food orally administered as drinking water or the like.

Example 7: Preparation of a pharmaceutical composition or an edible composition

150 mg of the glycolipid-containing composition of the present invention prepared by the above method, 125 mg of purified soybean oil, 15 mg of beeswax and vitamin ElOmg were heated to about 40 ° C under a nitrogen gas atmosphere, mixed well, and mixed homogeneous Things. This was supplied to a capsule filling machine to prepare a gelatin-coated capsule preparation having a capacity of 30 mg per grain. This preparation can be used as a pharmaceutical composition or an edible composition.

Industrial applicability

As described above, the glycolipid-containing composition of the present invention has useful physiological activities such as DNA synthase inhibitory activity, cancer cell growth inhibitory activity, and antitumor activity. It can be used as an anti-cancer agent (anticancer agent), and can be suitably used especially for functional foods.For example, it can be used as a functional food having an anti-cancer effect or a cancer-preventive effect. Pharmaceutical and edible compositions (food or food additives) It can also be used as an additive.

Claims

The scope of the claims

[I] Seaweed or terrestrial plant power is also purified, and at least monogalactosyldiasylglycerol (MGDG), digalatatosyldiasylglycerol (DGDG), and sulfoquinovosyldiasylglycerol (SQDG) are used as glycolipids. A glycolipid-containing composition to be contained.

[2] A glycolipid-containing composition obtained by subjecting the glycolipid-containing composition according to claim 1 to lipase treatment and containing at least monogalactosyl monoacylglycerol (MGMG) and sulfoquinovosylmonoacylglycerol (SQMG) as glycolipids. Composition.

[3] A DNA synthase inhibitor comprising the glycolipid-containing composition according to claim 1 or 2 as an active ingredient.

[4] An anticancer agent comprising the glycolipid-containing composition according to claim 1 or 2 as an active ingredient.

[5] A pharmaceutical composition comprising the glycolipid-containing composition according to claim 1 or 2 as an active ingredient.

[6] An edible composition comprising the glycolipid-containing composition according to claim 1 or 2.

[7] A sugar containing seagrass or a terrestrial plant as a raw material and at least glycolipid monosyldiacylglycerol (MGDG), digalatatosyldiasylglycerol (DGDG), and sulfoquinovosyldiasylglycerol (SQDG) as glycolipids A method for producing a lipid-containing composition.

[8] A lipase treatment of the glycolipid-containing composition produced by the method of claim 7, whereby at least glycolipid monogalactosyl monoacylglycerol (MGMG) and sulfoquinovosyl monoacylglycerol (SQMG) A method for producing a glycolipid-containing composition comprising

[9] The method according to claim 7, further comprising a step of purifying the glycolipid fraction by using a plant extract as a hydrophobic chromatography.

[10] The method according to claim 9, wherein the glycolipid is eluted with an alcohol such as ethanol or an organic solvent such as acetone, and the water-soluble substance is eluted with 50% to 75% of a water-containing organic solvent. A method comprising the step of eluting glycolipids with a 100% aqueous organic solvent or an organic solvent.

[II] The method according to claim 7, comprising a step of washing the plant with warm water at 40 ° C to 80 ° C to remove water-soluble components before obtaining the plant extract. [12] The method according to claim 7, wherein a green-yellow vegetable such as spinach (Spinacia) is used as a raw material.