JPH09301874A - Improving agent for cerebral function - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a cerebral function improving agent containing N- acetylneuraminic acid bonded oligosaccharides promoting the metabolism of cerebral phospholipid and gangliosides. SOLUTION: This agent contains the N-acetylneuraminic acid bonded oligosaccharides expressed by the formula (Gal)n-Neu5Ac (Gal is galactose residue; Neu5Ac is N-acetylneuraminic acid residue; (n) is 1 or 2), as the active ingredient. Other suitable conventional ingredients are blended and can be prepared in conventional method. This compound can be used in food and drink such as yogurt, cheese, bread, drinkable beverage, etc. The adult's dosage of the N-acetylneuraminic acid bonded oligosaccharides is at least 10mg/kg per day, preferably 30 to 100mg/kg per day. The N-acetylneuraminic acid bonded oligosaccharides is an oligosaccharides in which galactose is bonded with N- acetylneuraminic acid, obtained by glycosyltransfer reaction of β-galactosidase using lactose and N-acetylneuraminic acid or its salt as the raw material.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel cerebral function improving agent that promotes metabolism of phospholipids and gangliosides in the brain, and foods and drinks provided with such cerebral function improving action.

[0002]

2. Description of the Related Art Conventionally, gangliosides are considered to play some role in the nervous system because they are present in large amounts in the brain. The reason is that
(1) Ganglioside content in brain tissue is higher than that in any other tissue, and shows characteristic changes in the process of brain evolution and the process of brain tissue construction. In particular, it is known that the amount of ganglioside in brain tissue increases in infancy and decreases with aging (protein, nucleic acid, enzyme, vol.35, pp.535-545, 1990).

(2) Ganglioside promotes the release of neurotransmitters such as dopamine, serotonin and acetylcholine (Kanagi et al., Glycoconjugate, pp.124-135, published by Medical View Co., 1994). That is, since ganglioside has an action of promoting nerve differentiation and synaptic function,
A therapeutic effect for neurological disorders is expected. For example,
Parkinson's disease is a disease that causes muscular rigidity and a decrease in movement, and it is said that many elderly patients have dementia. As a model experiment for this Parkinson's disease, 1-methyl-4-phenyl- was added to mice.
When 1,2,3,6-tetrahydropyridine (MPTP) was injected to reduce the dopamine content to about 50% and then ganglioside was administered, recovery of dopamine content and improvement of behavior were observed. Has been revealed
(Hadjiconstantinou, M., J. Neurochem, vol.51, pp.11
90-1196, 1988). In addition, cerebral ischemic injury causes death and loss of neurons, resulting in loss of brain functions such as memory and intelligence, resulting in dementia. It has been reported that administration of gangliosides is effective against this disorder, and that administration of gangliosides improves cerebral edema and behavior and reduces mortality (Lombardi, G., Lett., Vo
l.134, pp.171-174, 1992).

(3) Gangliosides are said to act also in promoting brain synaptic function. That is, when synaptosomes prepared from rat brain are stimulated by high-potassium depolarization, transmitter release occurs. At this time, it is known that pretreatment of synaptosomes with ganglioside promotes the release of acetylcholine, which is a transmitter, (Bliss, TVP, Nature, vol.361, pp.31-39, 1993).

Sialic acid bound to this ganglioside is a general term for an acyl derivative of neuraminic acid, which is an acidic saccharide widely existing in the living world, and from lactose-6-phosphate to N in the living body of mammals. -Synthesized via acetylmannosamine. This sialic acid is particularly abundant in the brain and central nervous system as a constituent of gangliosides and glycoproteins, and the content of sialic acid rapidly increases during the growth period (infancy). It is thought to play an important role in the functional expression and development of. For example, Carlson et al. Have shown that oral administration of sialic acid to infant rats increases cerebral and cerebellar sialic acid content (Carlson, SE, J. Nutr., Vol. 116, p.
p.881-886, 1986). Morgan et al. Also reported that administration of sialic acid improved memory learning ability (Mor
gan, BLG, J. Nutr., vol.110, pp.416-424, 1980).
However, the increase in sialic acid content in the brain by oral administration of sialic acid is not high at all, and there is a demand for the development of a superior sialic acid compound which has an effect on the functional expression and development of brain and central nervous system tissues. .

On the other hand, phospholipids are important constituents of biological membranes and form a hydrophobic lipid bilayer membrane together with cholesterol. This lipid bilayer has various physiological functions as well as a mere septum. For example, in the context of nerve function, it is known that the addition of phospholipids to brain slices causes the release of acetylcholine. In addition, in experiments in which phospholipids were administered to animals, it was reported that improvement of memory impairment and restoration of acetylcholine releasing activity were observed in old age (Magil, SGet al.,
J. Nutr., Vol.111, p.166, 1981). Furthermore, phospholipids in the brain are known to decrease with age. It is believed that this phenomenon reduces the fluidity of the membrane, which in turn reduces the nerve conduction velocity (protein
Nucleic acid and enzyme, vol.35, p.527, 1990). Therefore, promoting metabolism of phospholipids in living tissues is considered to be very significant, since it also leads to increase in phospholipids in the brain.

[0007]

DISCLOSURE OF THE INVENTION The inventors of the present invention have been earnestly researching a substance having an effect of promoting an increase in phospholipid content or ganglioside content in the whole brain, and found that lactose and N-acetyl neu Using laminic acid as a raw material β
-General formula synthesized by the transglycosylation reaction of galactosidase
N-acetylneuraminic acid-linked oligosaccharide represented by (Gal) n-Neu5Ac (wherein Gal represents a galactose residue, Neu5Ac represents an N-acetylneuraminic acid residue, and n represents 1 or 2). Indicates an integer. The same applies below.)
Have the effect of promoting the metabolism of phospholipids and gangliosides in the whole brain, that is, having the effect of increasing the total amount of phospholipids and gangliosides in the brain.

Then, they have found that the brain function can be improved by administering this N-acetylneuraminic acid-linked oligosaccharide, and have completed the present invention.
Therefore, the present invention provides the general formula (Gal) n-Neu5A
It is an object of the present invention to provide a brain function improving agent containing an N-acetylneuraminic acid-linked oligosaccharide represented by c as an active ingredient. The present invention also provides a compound represented by the general formula (Gal) n-Neu5Ac.
It is an object of the present invention to provide a food or drink containing an N-acetylneuraminic acid-bonded oligosaccharide represented by the formula (1) to impart a brain function improving action.

[0009]

In the present invention, an N-acetylneuraminic acid-linked oligosaccharide represented by the general formula (Gal) n-Neu5Ac is used as an active ingredient. This oligosaccharide is an oligosaccharide in which galactose and N-acetylneuraminic acid are bound to each other, and is synthesized by lactose and N-acetylneuraminic acid or a salt thereof as a raw material by a transglycosylation reaction of β-galactosidase.

The N-acetylneuraminic acid-bonded oligosaccharide represented by the general formula (Gal) n-Neu5Ac includes oligosaccharides having the following structures. (1) O-β-D-galactopyranosyl- (1 → 8) -N
-Acetylneuraminic acid (2) O-β-D-galactopyranosyl- (1 → 9) -N
-Acetylneuraminic acid (3) O-β-D-galactopyranosyl- (1 → 3) -O
-Β-D-galactopyranosyl- (1 → 8) -N-acetylneuraminic acid

Sialic acid is generally located at the non-reducing end of sugar chain and is known to exert various physiological functions. However, the general formula (Gal) n used as the active ingredient of the present invention is known.
The sialic acid contained in the N-acetylneuraminic acid-bonded oligosaccharide represented by -Neu5Ac (hereinafter simply referred to as N-acetylneuraminic acid-bonded oligosaccharide) is characterized in that it is located at the reducing end of the sugar chain. . It should be noted that substances having such a sugar chain structure have only been reported to be present in rat embryonic nerve cells, and their physiological functions have attracted attention (Higuchi et al., Liebig.
S. Ann. Chem., pp. 1-10, 1991).

Next, the method for preparing the N-acetylneuraminic acid-bonded oligosaccharide as the active ingredient of the present invention will be specifically described.

[Reference Example 1] N- according to the description of JP-A-7-316177
Acetylneuraminic acid-linked oligosaccharide was produced. That is, lactose 1,000 g and sodium N-acetylneuraminate
After dissolving 560 g and warm water in 3,500 g and adjusting the pH to 6.0 with acetic acid, Bacillus circulan
500 mg of β-galactosidase (Yamato Kasei ) derived from s) was added and reacted at 40 ° C. for 48 hours. And this reaction solution is 1
Diameter that was filled with anion exchange resin (Dow 1; acetic acid type, Muromachi Kagaku) after stopping the enzymatic reaction by heating at 00 ℃ for 1 minute
The solution was passed through a column of 40 cm × 70 cm in length, and the produced N-acetylneuraminic acid-linked oligosaccharide and unreacted N-acetylneuraminic acid were adsorbed on the resin. Next, a sufficient amount of deionized water was passed through this column to remove galacto-oligosaccharides and other neutral sugars generated by the enzymatic reaction.
The N-acetylneuraminic acid-bonded oligosaccharide adsorbed on the resin was eluted by gradient elution using the sodium acetate solution in Example 1. Note that unreacted N
-It has become possible to completely separate -acetylneuraminic acid and N-acetylneuraminic acid-linked oligosaccharide. Further, the elution fraction containing the N-acetylneuraminic acid-bonded oligosaccharide was desalted by electrodialysis (model TS-24 type, membrane area: both cation membrane and anion membrane 960 dm ² , Tokuyama Corporation), and concentrated under reduced pressure. After freeze-drying, 250 g (purity: 95%) of white powder of N-acetylneuraminic acid-linked oligosaccharide was obtained.

The brain function improving action of the present invention will be described with reference to test examples.

[Test Example 1] Using the N-acetylneuraminic acid-bonded oligosaccharide obtained in Reference Example 1, changes in the amount of lipid in the whole brain were examined. As an experimental animal, an 8-week-old male SD rat (Charles River Japan) was used. First, after preliminarily raising all the rats with a standard diet (AIN-93 G) for 7 days,
The animals were divided into 4 groups each consisting of 6 animals, and the diets having the compositions shown in Table 1 were administered to each group.

[0014]

[Table 1] ─────────────────────────────────── Cont Lac NANA G-NANA ───── ────────────────────────────── α-cornstarch 13.2 13.2 13.2 13.2 cornstarch 39.7 39.7 39.7 39.7 milk casein 20.0 20.0 20.0 20.0 superior white sugar 10.0 6.0 6.0 6.0 Soybean oil 7.0 7.0 7.0 7.0 Crystalline cellulose powder 5.0 5.0 5.0 5.0 Mineral mixture ¹⁾ 3.5 3.5 3.5 3.5 Vitamin mixture ²⁾ 1.0 1.0 1.0 1.0 L-cystine 0.3 0.3 0.3 0.3 Choline bitartrate 0.25 0.25 0.25 0.25 tert-butyl Hydroquinone 0.0014 0.0014 0.0014 0.0014 Lactose − 4.0 − − N-acetylneuraminic acid − − 4.0 − N-acetylneuraminic acid-linked oligosaccharide − − − 4.0 ────────────────── ────────────────── Cont: Control group Lac: Lactose administration group NANA: N- Sechirunoiramin acid administration group G-NANA: N-acetylneuraminic acid linked oligosaccharides administration group ^{1): AIN-93-G} -MX-OYC 2): AIN-93-VX-OYC

The rats are bred at a humidity of 60%, a room temperature of 24 ° C. and a li.
The ght-dark control was performed for 12 hours, and the rats were fed with food and ion-exchanged water freely for 2 weeks. Then, after 2 weeks, the rat was anesthetized with ethyl ether, the whole brain was extracted, the weight of the whole brain was measured, and the whole brain was freeze-dried to analyze each lipid content in the whole brain. The results are shown in Table 2.

The analysis of each lipid in the whole brain was performed as follows. Extraction of total lipids To a whole brain sample (1 to 1.5 g) extracted from a rat, a solvent consisting of 30 volumes of chloroform: methanol: water (4: 8: 3) was added, homogenized for 5 minutes, and then centrifuged for 10 minutes. Separated to obtain an extract. In addition, the residue on centrifugation is chloroform:
30 ml of a solvent consisting of methanol: water (4: 8: 3) was added and re-extracted to obtain an extract. Then, combine both extracts 1
The volume was adjusted to 00 ml and used as a sample for lipid analysis.

Analysis of Ganglioside (1) Column Chromatography with Anion Exchange Resin 8 ml of DEAE-Sephadex A-25 (acetic acid type, Pharmacia) was packed in a column, and a double volume of chloroform: methanol: water (4: 8: 3) and then equilibrate with 50 ml of lipid extract.
The solution was passed through to adsorb acidic substances such as ganglioside.
Next, after elution of the non-adsorbed substance with 80 ml of a solvent consisting of chloroform: methanol: water (4: 8: 3), a solvent consisting of chloroform: methanol: 5M sodium acetate (30: 60: 8) 60
The acidic substance was eluted in ml and collected.

(2) Weak alkaline decomposition and dialysis After concentrating the eluate collected in (1) above, 20 ml of a methanol solution containing 0.5 M sodium hydroxide was added, and the mixture was allowed to stand at 37 ° C. for 2 hours to hydrolyze the ester lipid. Disassembled. Then, after neutralizing with acetic acid, methanol was removed, and mixed impurities were removed by dialysis. In addition, dialysis is performed at 5 ℃ for 2 days,
After completion of dialysis, the internal solution was concentrated and freeze-dried to obtain crude ganglioside.

(3) Silica gel column chromatography 2.5 g of iatro beads (Iatron Co., Ltd.) deaerated by suspending in a solvent consisting of chloroform: methanol (85:15) were filled in a glass column, and then chloroform: methanol (8
5:15) The crude ganglioside dissolved in 1 ml was passed through to adsorb the ganglioside. Next, after eluting impurities with 30 ml of a solvent consisting of chloroform: methanol (85:15),
Gangliosides were eluted with 50 ml of a solvent consisting of chloroform: methanol (3: 7). And after removing the solvent,
The volume was set to a fixed amount and used as a sample for quantification.

(4) Analysis of the amount of total sialic acid contained in ganglioside A fixed amount of a sample for quantification was taken, dried with nitrogen gas, and added with 2 ml of resorcinol-hydrochloric acid reagent, and the mixture was stirred.
Color was developed by heating at ℃ for 30 minutes. Then, after cooling immediately, 4 ml of a solution of butyl acetate: 1-butanol (85:15) was added to extract the dye, and the total sialic acid content in the ganglioside was determined by measuring the absorbance at 580 nm. It was quantified.

(5) Analysis of ganglioside composition A fixed amount of a sample for quantification was analyzed by high performance liquid chromatography.
It was injected into (HPLC) and the ganglioside composition was analyzed under the following measurement conditions. Column; Aquasil SS (6 mm x 200 mm) Eluent; Acetonitrile: Isopropyl alcohol: 50
mM tetraammonium chloride aqueous solution (20:68:12) ~ (5: 4
3:52) gradient elution detector; UV 208nm

Analysis of Lipids Triglyceride was analyzed using Triglyceride-Test Wako (acetylacetone method, Wako Pure Chemical Industries). In addition, the analysis of phospholipids was carried out using Phospholipid-Test Wako (Permanganate incineration method, Wako Pure Chemical Industries, Ltd.). In addition, the cholesterol analysis shows that the Determina TC555
(Enzymatic method, Kyowa Medix). The results are shown in Table 2.

[0023]

[Table 2] ─────────────────────────────────── Cont Lac NANA G-NANA ───── ────────────────────────────── Triglyceride 5.15 ± 0.83 4.91 ± 0.30 5.44 ± 0.53 3.92 ± 0.32 Cholesterol 9.52 ± 0.75 9.39 ± 0.48 10.00 ± 0.47 10.73 ± 1.08 Phospholipid 34.46 ± 1.66 34.13 ± 1.25 35.25 ± 1.56 36.73 ± 1.60 Ganglioside 0.89 ± 0.07 0.82 ± 0.05 0.93 ± 0.09 1.05 ± 0.05 ────────────────── ─────────────────── Cont: Control group Lac: Lactose administration group NANA: N-Acetylneuraminic acid administration group G-NANA: N-Acetylneuraminic acid binding oligo Sugar administration group

The values in the table are mean ± standard deviation, and the unit is mg / g whole brain wet weight. The phospholipid content and the ganglioside content in the whole brain were significantly increased in the N-acetylneuraminic acid-linked oligosaccharide-administered group as compared with the control group, the lactose-administered group and the N-acetylneuraminic acid-administered group. No changes were observed in the phospholipid fatty acid composition and the ganglioside composition in the whole brain.

[0025]

[Test Example 2] Using the N-acetylneuraminic acid-linked oligosaccharide obtained in Reference Example 1, a water maze experiment was conducted for the purpose of investigating the effect on the improvement of learning behavior due to the difference in ganglioside content in the whole brain. It was As an experimental animal, an 8-week-old male SD rat (Charles River Japan) was used. First, all rats were preliminarily bred with a standard diet (AIN-93 G) for 7 days, and then divided into 4 groups each consisting of 6 rats, and Test Example 1 was used.
The same feed composition as shown in Table 1 was administered to each group. Humidity of 60%, room temperature 24 ℃, light-da
The rk control was carried out under the condition of 12 hours, and the rats were allowed to freely ingest the feed and ion-exchanged water and bred for 10 days.

Then, a "water fille" as shown in FIG.
d multiple T-maze "with 120 vertical and horizontal lengths
The T-shaped maze is combined with an aquarium with a depth of 40 cm and a depth of 40 cm, and 11 blind lanes are placed, and the method of Ishizaki (Ishizaki, Exp. Ani
m., vol.27, pp.9-12, 1978) was carried out at a water temperature of 23-24 ℃. First, before the experiment, 5 trials were made in the straight waterway, and then 3 trials in the water maze for 4 consecutive days from the next day (total:
15 times), and the time required to reach the target point from the starting point of the water maze was measured. The result is shown in FIG.

On the 1st to 3rd day from the start of the water maze experiment, the time required to reach the target point from the starting point was significantly higher in the N-acetylneuraminic acid-linked oligosaccharide administration group than in the control group and the lactose administration group. It was short. In addition, the time required from the starting point to the target point on the second day of the water maze experiment was N in the N-acetylneuraminic acid-linked oligosaccharide administration group.
-It was significantly shorter than that of the acetylneuraminic acid administration group.

[0028]

BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, N-acetylneuraminic acid-linked oligosaccharide is used as an active ingredient of a brain function improving agent. This N-acetylneuraminic acid-bonded oligosaccharide is made into a preparation together with a carrier and other conventional components used for preparation or as it is. The dosage form is usually orally administered in the form of sugar-coated tablets, tablets such as tablets, granules, liquids, capsules and the like. Also, this N-
The acetylneuraminic acid-bound oligosaccharide may be used by blending it with a food or drink containing a nutritional composition and the like. Examples of such foods and drinks include yogurt, cheese, bread, drink beverages and the like. In addition, in order to promote the metabolism of phospholipids and gangliosides in the whole brain and exert a brain function improving effect, at least 10 mg / kg body weight per day of an adult, preferably 30 to 100 mg / kg body weight of N-acetylneuramine. It suffices to ingest acid-linked oligosaccharides.

[0029]

Example 1 1.1 g of N-acetylneuraminic acid-bonded oligosaccharide obtained in Reference Example 1 was filled in a gelatin capsule No. 000 for oral administration according to the Japanese Pharmacopoeia to prepare a brain function improving agent.

[0030]

Example 2 19.4 kg of warm water was added to 3 kg of skimmed milk powder, and the mixture was stirred, sterilized at 95 ° C for 10 minutes, and then cooled to 42 ° C. Lactobacillus bulgaricus (Lactobaci
llus bulugaricus) and Streptococcus thermophilus mixed starter MR
A culture was prepared by inoculating C-32 and fermenting at 42 ° C. for 4 hours. On the other hand, 5 kg of isomerized sugar, 125 g of pectin, and 110 g of N-acetylneuraminic acid-bonded oligosaccharide obtained in Reference Example 1 were added to 7.3 kg of water, dissolved by stirring, and sterilized at 90 ° C. for 10 minutes,
It cooled to 10 degreeC and prepared the sugar solution. Then, the above-mentioned culture was added to this sugar solution with stirring, homogeneously mixed, homogenized with a homogenizer and filled in a 1 liter-capacity paper yogurt to which a brain function improving effect was endowed. Manufactured.

[0031]

INDUSTRIAL APPLICABILITY N-acetylneuraminic acid-linked oligosaccharides have an effect of promoting metabolism of phospholipids and gangliosides in the whole brain, and show an effect of improving brain function such as improving learning behavior. It is useful as a material for products.

[Brief description of drawings]

FIG. 1 shows a plan view of a T-shaped water maze used in Test Example 2.

[Explanation of symbols]

 1-11 blind road number

FIG. 2 shows the time required for a rat to reach a target point from a starting point of a T-shaped water maze in Test Example 2.

Claims (3)

[Claims] 1. A brain function improving agent comprising an N-acetylneuraminic acid-linked oligosaccharide represented by the general formula (Gal) n-Neu5Ac as an active ingredient. (However, in the formula, Gal represents a galactose residue, Neu5Ac represents an N-acetylneuraminic acid residue, and n represents an integer of 1 or 2.) 2. A food or drink comprising an N-acetylneuraminic acid-linked oligosaccharide represented by the general formula (Gal) n-Neu5Ac to impart a brain function improving action. (However, the meaning of the formula is the same as above.) 3. An N-acetylneuraminic acid-bonded oligonucleic acid represented by the general formula (Gal) n-Neu5Ac produced by subjecting lactose and N-acetylneuraminic acid or a salt thereof to a transglycosylation reaction with β-galactosidase. The brain function improving agent or food or drink according to claim 1 or 2, which uses sugar. (However, the meaning of the formula is the same as above.)