JP2640971B2 - Glycosphingolipid - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to glycosphingolipids derived from Acanthaster planci and having a cerebral cortical neuronal survival maintaining effect.

[Conventional technology and its problems]

Glycosphingolipids are components of a fat bilayer that forms cell membranes of animals and plants, and are said to be involved in cell differentiation, mutual recognition, and the like.

Conventionally, several species of glycosphingolipids such as cerebroside (ceramide-monohexoside) ceramide-dihexoside and ganglioside have also been obtained from starfishes belonging to the phylum Echinoderm (T. Komori et al., Libeigs Ann. Chem, 1980,
653), both remain mixtures (molecular species) that differ in the composition of the ceramide (long-chain base and fatty acid) moiety. Structural analysis and bioactivity studies on the isolated glycosphingolipids have been conducted. Absent.

[Means for solving the problem]

The present inventors have reported that the sea star, Acanthas
ter planci), a novel ganglioside was isolated, and its structure was determined and its biological activity was examined. As a result, it was found that the novel ganglioside was represented by the following formula (I) and had a cerebral cortical nerve cell survival activity. The present invention has been completed.

That is, the present invention provides the following formula (I) (Wherein, R represents a linear alkyl group having 10 to 25, preferably 13 to 21 carbon atoms, and R 'represents a linear alkyl group having 10 to 20, preferably 11 to 17 carbon atoms) It provides glycolipids.

The glycosphingolipids (I) of the present invention can be prepared, for example, by treating a starfish with chloroform and alcohols (particularly methanol)
, Extracted with a mixed solvent, concentrated and then subjected to solvent fractionation (Example 1 described later).
And the water-soluble fraction is purified by reverse-phase and normal-phase column chromatography.

Typical examples of the glycosphingolipids of the present invention include AG-2-1 in which R = CH ₂ ) ₁₇ CH ₃ , R ′ = CH ₂ ) ₁₁ CH ₃ in the formula (I); Medium R = CH ₂ ) ₁₉ CH ₃ , R ′ =
AG-2-3 which is CH ₂ ) ₁₁ CH ₃ and R = CH in the formula (I)
_{2) 21 CH 3, R '} = CH 2) contains ₁₁ CH AG-2-5 is _3, which are separable by reverse phase high performance liquid chromatography (HPLC).

Next, the results of a test on the activity of the glycosphingolipid (I) of the present invention obtained as described above to maintain the survival of cerebral cortical neurons will be described.

MTT method: The cerebral cortex of a rat fetus (day 16-18) was dissociated with dispase and DNase and suspended in a serum-free medium. A 96-well multiplate coated with polyornithine was seeded at a density of 6400 cells / well (90 μ / well), and AG-2
-5 dissolved in DMSO and added (final concentration 100 μg / ml)
The cells were cultured in a carbon dioxide incubator for 20 hours. And
10 μM MTT ^* solution was added (final concentration 0.15 mg / ml) and the culture was continued for another 4 hours. 100μ of isopropanol-
0.08N HCl was added to stop the reaction.
Absorbance was measured using 90 nm as a reference wavelength. The results are shown in the table below.

As is clear from these results, the number of surviving cerebral cortical neurons decreases with time under normal culture conditions, but the addition of AG-2-5 protects the neurons and increases the number of surviving viable cells. Was.

〔The invention's effect〕

Since the glycosphingolipid (I) of the present invention has a cerebral cortical nerve cell survival maintaining effect as described above, it has use as a therapeutic drug for neurodegenerative patients such as senile dementia, Alzheimer's disease, Parkinson's disease and the like. Met.

〔Example〕

 Next, the present invention will be described in more detail with reference to examples.

Example 1. 52 kg of a chloroform-methanol extract was distributed in a mixed solution of water and ethyl acetate-n-butanol, and the water tank was concentrated to dryness and extracted with a mixed solution of chloroform-methanol. The chloroform-methanol soluble portion was purified by reversed-phase and normal-phase column chromatography to obtain 1.2 g of a component (AG-2) showing a single spot on normal-phase TLC.

Example 2. AG-2 obtained as described above showed amide absorption by IR, and ¹³ C NMR showed a signal specific to phytosphingosin-type ceramide having an oxyfatty acid. Also, from the ¹³ C NMR signal and the ion peak of negative fast atom bombardment mass spectrometry (FABMS), AG-2 was determined to be a phytosphingosin-type ganglioside molecular species having five sugars, and the sugar moiety structure was as follows. I made it clear.

First, from the findings of ¹³ C NMR, the results of methanolysis, TMS, and gas chromatography, the constituent sugars consisted of 1 mol each of N-acetylneuraminic acid (NANA), glucose (Glc), and 3 mol of galactose (Gal). From the fragmentation in the negative FABMS, it was found that terminal hexose → hexose → NANA → hexose → hexose.

Next, alditol obtained from the fully methylated form of AG-2
From GC-MS analysis of acetate and GC-MS analysis of acetate of completely methylated methanolyzate, terminal hexofuranose (1 mol), 3-position bound hexopyranose (1 mol), 4-position bound NANA (1 mol), 4-position bound The presence of hexopyranose (2 mol) was found.

Further, when AG-2 was heated at 80 ° C. in a pyridine-water mixture and partially hydrolyzed, ceramide-lactoside and oligosaccharide were obtained. This oligosaccharide was found to be Galf (1 → 3) Galp (1 → 3) based on the results of electrolytic desorption mass spectrometry (FDMS) of the completely methylated product and analysis of alditol-acetate.
4) It was a trisatsucaride made of NANA.

From the above chemical findings and the behavior of the signal of the anomeric carbon in ¹³ CNMR of AG-2, AG-2 is represented by the formula (I)
Was determined to be a mixture of compounds represented by

AG-2: mp.153-156 ° C. Negative FABMS (m / z) [1537,1565,1579,1593,1607
[MH]-] ¹³ C NMR [δ110.5 (d), 104.5 (d), 104.2 (d), 10
0.8 (s), 96.4 (d ) ] Example 3 Separation of components of AG-2 reverse phase HPLC [column using ^{_{MeOH-H 2 O-picA *}} : ERC-2151,3μ solvent: 97% MeOH-picA (1
00: 5) Flow rate: 1.2 ml / min] to isolate AG-2-5. And the structure of this product is ¹³ C NMR, negative FABM
S, identification of fatty acid methyl ester obtained by methanolysis, and molecular weight obtained by negative FABMS. ^* PicA (Io inhibitor; trade name) AG-2-1 and AG-2-3 were similarly isolated.

Claims (3)

(57) [Claims] 1. The following formula (I) (Wherein, R represents a straight-chain alkyl group having 10 to 25 carbon atoms, and R ′ represents a straight-chain alkyl group having 10 to 20 carbon atoms). 2. R is a straight-chain alkyl group having 13 to 21 carbon atoms;
Is a straight-chain alkyl group having 11 to 17 carbon atoms. [3] A therapeutic agent for neurodegenerative diseases, comprising the glycosphingolipid of claim 1 as an active ingredient.