JP3042738B2 - Ganglioside GM3 composition and method for producing the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to ganglioside G _M3 composition obtained Ganglioside G _D3 derived from milk by hydrolysis and a manufacturing method thereof. The ganglioside G _M3 composition obtained by the method of the present invention can be used for medicine, pharmacy, biochemistry,
Useful in fields such as food.

[0002]

2. Description of the Related Art Ganglioside is a general term for glycosphingolipids containing sialic acid, and their molecular species are diverse.
Among them, ganglioside G _M3 (hereinafter referred to as G _M3)
Has a structure in which lactose binds to ceramide and sialic acid has an α2-3 bond structure at its non-reducing end. Further, (hereinafter abbreviated as G _D3) Ganglioside G _D3 is further sialic acid to the non-reducing end of the G _M3 contains two molecules of sialic acid in the form of a binding Arufa2-8. Sialidase is an enzyme that cleaves sialic acid. When the action of sialidase against G _D3 sialic acid released from G _D3, ultimately, would be degraded to lactosylceramide. _GM3 is believed to play a variety of roles in vivo, such as regulating the phosphorylation of EGF receptors, acting as a receptor for influenza and Newcastle disease virus, and regulating blood cell differentiation. Or

[0003] Glycosphingolipids including gangliosides are composed of a sugar chain part and a lipid part called ceramide as described above, and the ceramide part is composed of a long-chain base and a fatty acid. Glycosphingolipids are generally classified according to their sugar chain portions, but it is known that the ceramide portion is classified into the same glycolipid and has the same sugar chain but has diversity. In other words, the same glycolipid derived from the same tissue has the same sugar chain but is composed of various long-chain bases and fatty acids. It is also known that this diversity is characterized by organs and tissues containing glycosphingolipids.

[0004] It is also known that the physiological function of glycosphingolipids is carried by the sugar chain portion. On the other hand, the fact that the structure of the ceramide portion is greatly involved in the expression of the physiological function is also known as the anti-stress ulcer activity cerebroside. Etc. are known. It was said to be a receptor when an influenza virus G _M3, more chain length fatty acids in the ceramides long, it is also known that high affinity between the influenza virus and G _M3.
In other words, the expression of physiological functions of glycosphingolipids is affected not only by the sugar chain structure but also by the structure of the ceramide moiety.

[0005] The structure of G _M3, when the milk-derived G _M3, long chain base sphingosine (d18: 1), fatty acids Palmitic acid (C16: 0) and Stearic acid (C18: 0) Oleic acid (C18: It is known that 1) is a major constituent fatty acid (J. Biol. Chem. 261, 5625-5630 (1985)). Also,
Although G _M3 is general that are isolated and purified from bovine brain or cow's breast, the fatty acid composition of the G _M3 is not much different from that derived from milk. Currently commercially available _GM3 is isolated and purified from bovine brain or cow's udder, but is insufficient from the viewpoint of infection protection due to the short chain length of constituent fatty acids.

[0006]

DISCLOSURE OF THE INVENTION The present inventors have studied the chemical structure and physiological activity of various naturally and artificially obtained gangliosides. Particularly G _D3 derived from milk by the action of sialidase or an acid, where only the non-reducing terminal sialic acids per molecule of sialic acid moiety ganglioside G _D3 with desialylated and G _M3, was studied constituents of the ceramide, Fatty acids are higher than conventional 16-18 carbon atoms,
G containing a large amount of long-chain fatty acids composed of 20 to 24 carbon atoms
_{An M3} composition has been found. Then, were examined the physiological activities of the G _M3 composition, and found that than G _M3 isolated from conventional bovine brain or cow udder of infection protective effects superior to influenza virus or the like, the present invention I've reached the point. An object of the present invention, with respect to such influenza viruses and the like, excellent have protective effect against infections, and to provide a good G _M3 ganglioside composition and their preparation of the physiologically active.

[0007]

SUMMARY OF THE INVENTION The present invention relates to a ganglioside represented by the general formula (5) obtained from milk or dairy products. General formula Neu5Acα2 → 3Galβ1 → 4Glcβ1 → 1C
er (5) (where Neu represents neuraminic acid, Gal represents galactose, Glc represents glucose, and Cer represents ceramide). The long-chain bases and fatty acids of the main constituents of the ceramide are composed of various long-chain bases and fatty acids represented by the general formulas (1), (2), (3) and (4).

[0008]

(Equation 5)

(Equation 6)

Equation 7

(Equation 8)

That is, the main constituent components of ceramide are long-chain bases such as sphingosine (d18: 1) (1) and hexadecadesphingenine (d1).
6: 1) (2), sphinganine (d18: 0) (3),
Hexsadecasphinganine, d
16: 0) (4) and the fatty acid is docosanoic acid (docosanoic aci
d, C22: 0), tricosanoic acid (C23:
0), tetracosanoic acid (C24:
0), tetracosenoic acid (C24: 1)
And the ratio of the fatty acids is C22: 0), C23: 0, C24: 0, C
The present invention relates to a ganglioside _GM3 composition, which is 20 to 40%, 15 to 35%, 10 to 30%, and 5 to 15% in the order of 24: 1. A feature of the present invention is that such a ganglioside _GM3 composition contains a large amount of the above-mentioned long-chain fatty acid having 22 to 24 carbon atoms as a constituent fatty acid of the ceramide.

[0010] To obtain Ganglioside G _M3 compositions of the invention have been known to be prepared in quantity ganglioside G _D3 composition from milk (see JP-A-63-269992), ganglioside in this way the G _D3 composition is prepared, can be obtained by desialylated by or acid exerts a sialidase this G _D3 composition. In addition, whey protein concentrate (WPC),
Ganglioside G _D3 prepared from buttermilk, etc., may be using the same.

Furthermore, milk or milk containing the ganglioside G _M3 composition of the present invention can be prepared by directly acting sialidase or acid on milk or milk products such as WPC and buttermilk without using the ganglioside G _D3 composition as a starting material. You can also get products. In the method using the sialidase, and ganglioside G _D3 compositions desialylated, suspended in desialylated sialidase buffer solution for performing, or, the dissolved Ganglioside G _M3 compositions performed sialic acid enzymatic degradation reaction The ganglioside _GD3 composition to be obtained or desialylated is suspended or dissolved in a buffer solution having an appropriate pH, and sialidase is acted thereon to hydrolyze sialic acid to obtain a ganglioside _GM3 composition.
Sialidase used in the present invention is derived from Vibrio cholerae,
A ganglioside _GM3 composition can be synthesized using any commercially available sialidase, such as one derived from Clostridium and one derived from Arthrobacter. The buffer solution used may be any buffer solution such as an acetate buffer, a phosphate buffer, a citrate buffer, and a maleate buffer, as long as the pH allows the enzyme to be used. Buffer concentration is 20-
Good between 200mM. In addition, the substrate ganglioside G _D3
The concentration of the composition is preferably 0.5 to 5 mg per ml. Regarding the amount of enzyme, if the amount is too large, lactosylceramide is decomposed, and if the amount is small, the reaction does not proceed. Therefore, the amount of enzyme is 2 to 20 muni per 1 mg of substrate.
Good during t. In the method using sialidase in the present invention, it is desirable to prepare three conditions of a buffer solution concentration, a substrate concentration, and an enzyme amount.

On the other hand, in the hydrolysis method using an acid, the relationship between the pH of the reaction solution, the reaction temperature and the time is very important. Due to the low pH and high reaction temperature, G
The formation rate of _M3 increases, but the decomposition rate also increases.
It is necessary to set the reaction time according to the conditions of pH 2 to 5 and reaction temperature 37 ° C to 100 ° C. Using the method of the present invention, relatively inexpensive and mass-ganglioside G _D3 compositions available, it can be prepared easily and in large quantities Ganglioside G _M3 composition. The structure of the ganglioside obtained by the method of the present invention was analyzed by the following method.

1. Proton nuclear magnetic resonance spectrum According to a known method, about 3 mg of ganglioside G _M3 prepared from milk-derived ganglioside G _D3 was replaced with heavy water, and then dissolved in dimethyl sulfoxide-heavy water (98: 2).
The proton nuclear magnetic resonance spectrum was measured at 90 ° C. FIG. 4 shows the obtained spectrum. In addition, when the ganglioside G _M3 obtained in Example 1 was measured by the same method using the proton magnetic resonance spectrum, it was found to be consistent with FIG. From these results, it was proved spectroscopically that the sugar chain structure of the ganglioside of the present invention was as shown in the formula (5).

2. Determination of Sialic Acid by Methylation Analysis Ganglioside G _M3 prepared by the method of Example 1 from about 1 mg of ganglioside G _D3 derived from milk was completely methylated by the method of Hakomori, and half of it was treated with 0.5 ml of 0.3N methanolic hydrochloric acid 0.3N. Perform methanolysis at 75 ° C for 18 hours. After distilling off the solvent, the residue was dried and trimethylsilylated (hereinafter referred to as TMS) at 60 ° C. for 20 minutes. This TMS derivative was analyzed by GLC / MS (GLC column was DB-1, MS ionization voltage was 7).
0 eV). From the results, N-methyl-4,7,8,9-tetra-O-methyl-N-acetyl-neuraminylmethylketoside methyl ester (N-Methyl-4,7,8,9-tetra-Om
ethyl-N-acetyl-neuraminylmethylketoside methyl est
er) was confirmed.

3. Determination of Sugar Chain Bonding Position by Methylation Analysis Using half of the fully methylated ganglioside G _M3 of the above 2,
A partially methylated alditol acetate derivative was prepared according to a known method. GLC / MS analysis (GLC column was DB-1, MS ionization voltage was 70 eV) showed that 1,3,5-tri-acetyl-2,4,6-tri-methyl-galactitol (1,3,3). 5-tri-acetyl-2, 4, 6-tri-
methly-garactitol) and 1,4,5-tri-acetyl-2,
3,6-tri-methyl-glucitol (1,4,5-tri-mety
l-2, 3, 6-tri-methly-gluctitol).

4. Constituent fatty acids Approximately 1 mg of ganglioside G _M3 prepared from milk-derived ganglioside G _D3 by the method of Example 1 was placed in a screw-mouth test tube with a Teflon seal, and 1 ml of aqueous methanolic 1
N hydrochloric acid was added, and the mixture was heated at 75 ° C for 18 hours. After cooling, the mixture was extracted three times with 2 ml of hexane to obtain a fatty acid methyl ester. Further, in order to acetylate the hydroxy acid, the solvent was removed with nitrogen, and the mixture was dried well, and 0.1 ml of pyridine-acetic anhydride (1: 1) was added thereto, followed by heating at 80 ° C. for 30 minutes. The obtained derivative was analyzed by GLC / MS (DB-1). Result is,
As shown in Table 1.

[0017]

[Table 1] No hydroxy acid was detected. tr. Indicates a trace, and n. d. Indicates not detected.

5. Constituent long-chain base The methanol solution obtained by extracting with hexane in the step 4 is dried in a nitrogen stream, a trimethylsilylating agent is added, and the mixture is heated at 60 ° C for 20 minutes. The obtained derivative was analyzed by GLC / MS. (G
LC column is DB-1, MS ionization voltage is 70e
V) The results are as shown in Table 2.

[0019]

[Table 2]

From the properties shown in the above 1 to 5, the ganglioside according to the present invention has the formulas (1), (2), (3) and (4): Neu5Acα2 → 3Galβ1 → 4Glcβ1 → 1C
er.

[0021]

According to the present invention, a novel ganglioside _GM3 composition having a different molecular species can be obtained, and the novel ganglioside _GM3 composition has a physiological activity that greatly improves the ability to protect against infection. Further, the ganglioside G _M3 obtained has been difficult up to now can be obtained easily and a large amount. Therefore, it is very useful in the fields of medicine, pharmacy, biochemistry, foods, etc., and uses the ganglioside _GM3 composition of the present invention to provide functional foods, high-bioactive infant milk powder, medicines, reagents, etc. Can be manufactured.

[0022]

The present invention will be specifically described below with reference to examples. However, the thin layer chromatography used in the examples was performed using Merck No. 13748, and the developing solvent was chloroform: methanol: 0.2% CaCl ₂ aqueous solution = 5.
At 5:45:10, orcinol coloring and resorcinol coloring were used.

Example 1 1 g of ganglioside G _D3 (derived from milk) and 5 units of sialidase (derived from Arthrobacter) were suspended in 500 ml of a 0.1 M acetate buffer (pH 7.0). And immediately the reaction temperature
The reaction was performed at 40 ° C. for 24 hours. During the reaction using thin layer chromatography confirmed the production of ganglioside G _M3. The thin-layer chromatography at the stage where the reaction was stopped is shown in FIG. After completion of the reaction, the enzyme was inactivated by leaving it in boiling water for 1 minute. Subsequently, the white powder obtained by drying under reduced pressure was subjected to ion exchange chromatography using DEAE-SephadexA-25 (Pharmacia).
Silica gel chromatography using Iatrobeads 6RS 8060 (Yatron) was performed to separate reaction products. The obtained reaction product was freeze-dried to obtain 415 mg of a white powder.

When this powder was analyzed by thin-layer chromatography, it was found to have the same Rf value as that of the standard product G _M3 (from bovine brain) (see FIG. 2). In addition, this powder is NM
Analysis by R and IR showed good agreement with the standard product (see FIGS. 3 and 4). These results white powder was obtained, it was confirmed that the ganglioside G _M3.
Ganglioside G _M3 obtained here, hydrochloric acid - with methanol and the free fatty acids, was measured fatty acid composition using GC-MS, different fatty acid composition than the ganglioside G _M3 was known conventionally, Table 1 shows the fatty acid composition. Incidentally, the fatty acid composition of Ganglioside G _M3 having different origin for reference are shown in Table 3.

[0025]

[Table 3]

Example 2 500 mg of ganglioside G _D3 (from milk) and 5 units of sialidase (from Streptococcus) were suspended in 2 liters of 50 mM maleic acid buffer (pH 5.0). Then, the reaction was immediately performed at a reaction temperature of 37 ° C. for 12 hours. After completion of the reaction, the enzyme was inactivated by leaving it in boiling water for 1 minute. Subsequently, the same treatment as in Example 1 was performed, and the reaction product was converted into a white powder.
mg was obtained. When the obtained powder was analyzed by the same method as in Example 1, it was confirmed that the powder was the ganglioside _GM3 composition of the present invention.

Example 3 10% containing 40 mg of ganglioside G _D3 per liter
Hydrochloric acid is added to the whey protein concentrated solution (WPC solution) to adjust the pH.
It was adjusted to 2.5 and reacted at a reaction temperature of 37 ° C. for 8 hours. After completion of the reaction, neutralization was performed using sodium hydroxide. After extracting the lipid fraction from this solution, thin layer chromatography analysis was performed.
It was confirmed that 10 mg of the ganglioside _GM3 composition of the present invention was contained.

Example 4 10% containing 28 mg of ganglioside G _D3 per liter
The buttermilk solution was adjusted to pH 4 by adding citric acid and reacted at a reaction temperature of 100 ° C. for 15 minutes. After completion of the reaction, the mixture was neutralized with sodium hydroxide and analyzed by thin-layer chromatography in the same manner as in Example 3. As a result, it was found that the ganglioside _GM3 composition of the present invention contained 46 mg per liter in the solution. all right.

[0029] Ganglioside G _M3 compositions and commercially available gangliosides G _M3 of the present invention obtained in Example 5 Example 1, by using the G _D3 derived from milk measured receptor recognition specificity of influenza virus. The measuring method was the method of Suzuki et al. (J. Biol. Chem., 260, 136).
2-1365 (1985)). Table 4 shows the results.
The results showed that the _GM3 composition obtained in the present invention had higher recognition specificity of influenza virus than the conventional product. This indicates that the ganglioside G _M3
The composition shows that the ability to protect against infection is higher than conventional _GM3 .

[0030]

[Table 4] Recognition of ganglioside by influenza ──────────────────────── Recognition specificity of ganglioside ───────────── ─────────── G _M3 (example 1) 21 ± 4 G _M3 (commercially available) 13 ± 3 G _D3 (derived from milk) 0 ───────────── ─────────── (The unit is nmol / mG protein / min.)

[Brief description of the drawings]

FIG. 1 shows the results obtained by thin-layer chromatography at the stage when the reaction was stopped in Example 1.

FIG. 2 shows a ganglioside G of the present invention obtained in Example 1.
It is the result obtained by thin-layer chromatography of _M3 and _GM3 as a standard.

FIG. 3 shows IR of ganglioside G _M3 obtained in Example 1.
It is a spectrum.

FIG. 4 shows the NM of ganglioside G _M3 obtained in Example 1.
It is an R spectrum.

────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Eiji Eiji 71-6-6-2026 Irumagawa, Sayama City, Saitama Prefecture (56) References JP-A-3-219841 (JP, A) Masahisa Maeno, Masayoshi Ohjo “ Dairy Technology Library, Theory of Milk Processing Method (Augmented 4th Edition) "(Showa 31-7-20), Asakura Shoten, pp. 404-406 (58) Fields investigated (Int. Cl. ⁷ , DB name) C07H 15/10 A61K 31/7032 A61P 31/12

Claims (4)

(57) [Claims] 1. A milk-derived ganglioside G _D3 is obtained by hydrolysis, and its main composition is represented by the following general formulas (1), (2), (3) and
Ganglioside G _M3 composition gangliosides G _M3 mixtures represented by (4). (Equation 1) (Equation 2) (Equation 3) (Equation 4) 2. The ratio of fatty acids of the main composition of the ceramide moiety is 20-40% of docosanoic acid (C22: 0), and tricosanoic acid.
(C23: 0) 15-35%, tetracosanoic acid (C24: 0) 10-30
The composition of claim 1 which is a ganglioside G _M3 mixture consisting of 5% to 15% tetracosanoic acid (C24: 1). Wherein only sialic acid per molecule of the non-reducing end by the action of sialidase to ganglioside G _D3 derived from milk and hydrolyzed to desialylated claim 1, wherein the obtaining Ganglioside G _M3 mixture ganglioside G of
Method for producing _M3 composition. 4. The ganglioside G _M3 mixture according to claim 1 , wherein an acid acts on the ganglioside G _D3 derived from milk to hydrolyze only one molecule of sialic acid at the non-reducing end to desialylate it. A method for producing a ganglioside G _M3 composition of the invention.