JP2997419B2 - Brain ganglioside content decrease inhibitor - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a brain ganglioside content decrease inhibitor which suppresses a decrease in brain ganglioside content, particularly a brain ganglioside content decrease with aging, and to a food or drink having the action.

[0002]

2. Description of the Related Art Ganglioside is a general term for glycolipids having sialic acid in the sugar chain portion, and is located on the surface of cells and performs various physiological functions such as recognition between cells, differentiation and induction of cells. It is believed that. In addition, it has a neutralizing effect on cholera toxin (Fishman, Journa
l of Membrane Biology, vol. 69, pp. 85-97, 1982), having a neutralizing effect on toxins produced by Clostridium botulinum (Kitamura, Biochemicaet Biophisica, vol. 628, pp.
328-335, 1980), and has a neutralizing effect on toxins produced by tetanus (Rogers and Synder, Journal of Bio
logical Chemistry, vol. 255, pp. 2402-2407, 1981). Furthermore, it is also known to exert receptor functions for various hormones, interferons and the like in vivo.

Heretofore, gangliosides are considered to play some role in the nervous system because they are present in a large amount in the brain. The reasons are (1)
The ganglioside content in brain tissue is higher than that in any other tissue, and shows characteristic changes in the process of brain evolution and in the construction of brain tissue. In particular, it is known that the ganglioside content in brain tissue increases in infancy and decreases with aging (proteins, nucleic acids, enzymes, vol. 35, pp. 535-545, 1990).

(2) Gangliosides promote the release of neurotransmitters such as dopamine, serotonin, and acetylcholine (Kanaki et al., Glycoconjugates, pp. 124-135, published by Medical View, 1994). In other words, gangliosides have the effect of promoting neural differentiation and synaptic function,
A therapeutic effect on neuropathy is expected. For example,
Parkinson's disease is a disease that results in muscle stiffness and loss of movement, and it is said that many elderly patients have the possibility of dementia. As a model experiment for this Parkinson's disease, 1-methyl-4-phenyl-
After injecting 1,2,3,6-tetrahydropyridine (MPTP) and reducing the amount of dopamine to about 50%, and then administering ganglioside, it is clear that the recovery of dopamine amount and improvement of behavior are observed. (Hadji
constantinou, M., J. Neurochem, vol.51, pp.1190-119
6, 1988). Also, cerebral ischemic injury results in death and loss of neurons, resulting in loss of brain functions such as memory and intelligence, resulting in dementia. Ganglioside administration is also effective for this disorder, and it has been reported that administration of ganglioside improved brain edema and behavior and reduced mortality (Lombardi, G., Lett., Vol. 134, vol. p
p. 171-174, 1992).

(3) Gangliosides are also said to work on promoting brain synaptic functions. That is, when synaptosomes prepared from rat brain are depolarized and stimulated with high potassium, a transmitter is released. At this time, it is known that pre-treatment of synaptosomes with ganglioside promotes release of acetylcholine, a transmitter.
(Bliss, TVP, Nature, vol. 361, pp. 31-39, 1993).

[0006] In addition, at least 3,000 or more cases of administration of ganglioside to humans have been reported by 1993. For example, it is known that by administering ganglioside to diabetic peripheral neuropathy patients, a certain improvement effect is exhibited (Eduardo, Drugs, vol.4).
7, pp.576-585, 1994; Bradley Muscleand Nerve, vol.
13, pp.833-842). Thus, clinical cases using gangliosides are expected to increase in the future, but this ganglioside is a very small component in the biological world, and it is difficult to extract and purify, so the price is high. There's a problem. Therefore, it has been desired to find a substance having the same effect as ganglioside or a substance having an action of promoting an increase in ganglioside content in a living body.

On the other hand, sialyl lactose is a sialic acid compound in which N-acetylneuraminic acid is α2-3 or α2-6 bonded to a galactose residue of lactose,
It is known that it is contained in trace amounts in milk and dairy products. In recent years, the physiological effects of this sialyl lactose have been attracting attention. For example, sialyl lactose in which N-acetylneuraminic acid is α2-3 linked to a galactose residue of lactose is Campylobacter pylori, which is said to be a causative agent of gastritis. Inhibits adhesion of (Campylobacter pylori ) to mucous membranes (Infect Immun., Vol. 56, pp. 2896-2906, 19
88) S, the causative agent of meningitis and sepsis in neonates
-Inhibiting the adhesion of E. coli (Acta Paediatr, vol.
82, pp. 6-11, 1993). Also, N-acetylneuraminic acid is added to the galactose residue of lactose by α2-6
Bound sialyl lactose is known as a receptor for influenza A virus (Nature, vol. 3).
33, pp. 426-431, 1988).

[0008] The effects of these sialic acid compounds on the brain have been studied for N-acetylneuraminic acid. That is, when N-acetylneuraminic acid is orally administered to an infant rat, N-acetylneuraminic acid is expressed in the cerebrum and cerebellum.
Increased acetylneuraminic acid content (Carlson, S. et al.
E., J. Nutr., Vol. 116, pp. 881-886, 1986) and improving memory and learning ability (Morgan, BLG, J. Nutr., Vol.
110, pp. 416-424, 1980). Further, the effect of sialyl lactose on the brain has been studied in the invention relating to milk powders containing sialyl lactose derived from milk. That is, when sialyl lactose derived from milk is administered to a rat in infancy, the sialic acid content in the brain reaches a certain value at an early stage (Japanese Patent Publication No. 63-65285). In neonates, since the ability to synthesize N-acetylneuraminic acid in the liver is not yet developed, administration of milk-derived sialyl lactose at this time is considered to be significant. However, in the present invention,
No mention was made of the relationship between the sialic acid (ganglioside) content in the brain and the learning function, and the subjects who took milk-derived sialyllactose were infants. It is known that the content of sialic acid (ganglioside) in the brain becomes constant in adulthood and then gradually decreases with age (Hyundai Kagaku Shonen 24, Aging Science, Tokyo Chemical Dojin, 1994). . Since the decrease in the sialic acid (ganglioside) content in the brain affects the functions of the brain and the nervous system, it is important how the decrease is moderated. Therefore, there has been a strong demand for the development of a substance having an action of suppressing a decrease in sialic acid (ganglioside) content in the brain.

[0009]

DISCLOSURE OF THE INVENTION The present inventors have intensively studied a substance that suppresses a decrease in brain ganglioside content and promotes an improvement in brain function. The present inventors have found that sialyl lactose suppresses a decrease in brain ganglioside content and has an effect of improving learning behavior, thereby completing the present invention. Therefore, an object of the present invention is to provide a medicament for suppressing a decrease in brain ganglioside content using sialyl lactose or a salt thereof as an active ingredient. Another object of the present invention is to provide a food or drink having a cerebral function improving effect by adding sialyl lactose or a salt thereof, thereby imparting a brain ganglioside content reduction suppressing effect. An object of the present invention is to improve the brain function particularly with the aging of the elderly or the elderly.

[0010]

According to the present invention, sialyl lactose or a salt thereof is used as an active ingredient. As sialyl lactose, milk-derived sialyl lactose or salts thereof are desirable. Milk-derived sialyl lactose can be obtained, for example, by a method for separating sialic acid-containing oligosaccharides (Japanese Patent Laid-Open No.
-79800). That is, whey discharged during the production of cheese is treated with an ultrafiltration membrane to remove proteins,
This is treated with a simulated moving bed chromatograph (SMB) to obtain a fraction containing about 5% by weight of sialyl lactose. Then, this fraction was concentrated with a rotary evaporator, sialyl lactose was adsorbed on an anion exchange resin, and neutral sugar was eluted with deionized water.
The adsorbed sialyl lactose is eluted with sodium acetate by a gradient method, further desalted by electrodialysis, concentrated under reduced pressure, and freeze-dried to obtain high-purity sialyl lactose. Sialyl lactose derived from bovine colostrum and sialyl lactose derived from human milk are commercially available.

Next, a method for producing sialyl lactose will be specifically described.

[Reference Example 1] Whey discharged during cheese production 1,5
100 liters were passed through an ultrafiltration membrane (Cefilt, fractional molecular weight 10 kDa,
1.4m ² membrane area, manufactured by NG Filtec) to remove proteins and remove lactose crystals by seeding.
After concentrating the obtained lactose crystal mother liquor to a concentration of 30% using an evaporator, a simulated moving bed chromatographic separator (SMB)
To give a treatment solution. The eluent used was deionized water, and the SMB was 8 mm with a column diameter of 25 mm and a length of 460 mm.
Each column was filled with a counter ion of a cation exchange resin UBK510L (Mitsubishi Chemical) as a Na type. The operating conditions of the SMB were as follows: processing solution supply rate 3.4 ml / min, eluent supply rate 5.8 ml / mi
n, the amount of raffinate withdrawn was 4.2 ml / min, the amount of extract was 5.0 ml / min, the column temperature was 10 ° C., and the step time was 7.40 minutes. Thus, the lactose crystal mother liquor concentrate
When 31 liters were treated, 38 liters of a non-acidic sugar fraction containing lactose and the like in the extract and 46 liters of a fraction containing sialyl lactose in the raffinate were obtained.

The raffinate was concentrated by an evaporator and passed through an anion exchange resin (Dow 1, acetic acid type) column having a diameter of 40 cm × 70 cm to adsorb sialyl lactose. After neutral water was eluted by passing a sufficient amount of water, sialyl lactose adsorbed with 0 to 0.06 M sodium acetate was eluted with a gradient. Under these elution conditions, sialyl lactose could be completely separated and recovered. Further, the obtained sialyl lactose eluate was desalted, concentrated, and freeze-dried to obtain 480 g of sialyl lactose white powder. When the purity of this sialyl lactose powder was measured by high performance liquid chromatography, the purity was 97% or more.

The effect of the sialyl lactose thus obtained on inhibiting the reduction of brain ganglioside content was as follows.

Test Example 1 The sialyl lactose obtained in Reference Example 1 was used to examine the effect of suppressing the decrease in brain ganglioside content. As an experimental animal, an 8-week-old male SD rat (Charles River Japan) was used. First, all rats were preliminarily reared for 7 days on a standard diet (AIN-93G), and then divided into four groups each consisting of six animals, and each group was administered a feed having the composition shown in Table 1.

[0014]

[Table 1] ──────────────────────────────────── Cont Lac NANA SL ────── ────────────────────────────── α-corn starch 13.2 13.2 13.2 13.2 corn starch 39.7 39.7 39.7 39.7 milk casein 20.0 20.0 20.0 20.0 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 Tertiary butyl Hydroquinone 0.0014 0.0014 0.0014 0.0014 Lactose-4.0--N-acetylneuraminic acid--4.0-Sialyl lactose---4.0 ──────────────────────── ──────────── Cont: control group Lac: lactose administration group NANA: N-acetylneura Phosphate administration group SL: sialyllactose administration group ^{1): AIN-93-G} -MX-OYC 2): AIN-93-VX-OYC

Rats are kept at a humidity of 60%, a room temperature of 24 ° C., and
The ght-dark control was performed for 12 hours, and the rats were fed with food and ion-exchanged water freely for 2 weeks. Two weeks later, the rats were anesthetized with ethyl ether, the whole brain was excised, the weight of the brain was measured, and then lyophilized to analyze the content of each lipid in the brain.

The analysis of each lipid in the brain was performed as follows. Extraction of total lipids A 30-fold amount of a solvent consisting of chloroform: methanol: water (4: 8: 3) was added to a brain sample (1 to 1.5 g) extracted from a rat, homogenized for 5 minutes, and then centrifuged for 10 minutes Thus, an extract was obtained. Further, 30 ml of a solvent consisting of chloroform: methanol: water (4: 8: 3) was added to the residue of the centrifugation, followed by re-extraction to obtain an extract. Then, combine these two extracts for 100
The volume was made up to ml, and this was 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 twice the amount of chloroform: methanol: water (4: 8: 3) After equilibration, 50 ml of lipid extract
Then, acidic substances such as ganglioside were adsorbed.
Next, the non-adsorbed substance was eluted with 80 ml of a solvent composed of chloroform: methanol: water (4: 8: 3), and then a solvent composed of chloroform: methanol: 5M sodium acetate (30: 60: 8) was eluted.
The acidic substance was eluted and collected in ml.

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

(3) Silica gel column chromatography A glass column was filled with 2.5 g of deaerated Iatrobeads (Iatron) suspended in a solvent composed of chloroform: methanol (85:15), and then charged into a chloroform column.
5:15) Crude ganglioside dissolved in 1 ml was passed through to adsorb ganglioside. Next, elute impurities with 30 ml of a solvent composed of chloroform: methanol (85:15),
Ganglioside was eluted with 50 ml of a solvent consisting of chloroform: methanol (3: 7). And after removing the solvent,
The volume was fixed to a fixed amount to obtain a sample for quantitative determination.

(4) Analysis of the total amount of sialic acid contained in ganglioside A fixed amount was taken from a sample for quantification, dried with nitrogen gas, and 2 ml of resorcinol hydrochloride reagent was added, followed by stirring.
Heat at 30 ° C. for 30 minutes to develop color. After cooling immediately, 4 ml of a solution consisting of butyl acetate: 1-butanol (85:15) was added to extract the dye, and the absorbance at 580 nm was measured to determine the total amount of sialic acid contained in the ganglioside. did.

(5) Analysis of ganglioside composition A high-performance liquid chromatograph of a fixed amount from a sample for quantification
(HPLC) and the ganglioside composition was analyzed under the following measurement conditions. Column; Aquasil SS (6mm × 200mm) Eluent; Acetonitrile: isopropyl alcohol: 50
mM tetraammonium chloride aqueous solution (20:68:12) to (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, Ltd.). The analysis of phospholipids was performed using phospholipid-Test Wako (permanganate incineration method, Wako Pure Chemical Industries, Ltd.). In addition, analysis of cholesterol revealed that
(Enzyme method, Kyowa Medex). Table 2 shows the results.

[0023]

[Table 2] ──────────────────────────────────── Cont Lac NANA SL ────── ────────────────────────────── Triglyceride 5.15 ± 0.83 4.91 ± 0.30 5.44 ± 0.53 5.53 ± 0.91 Cholesterol 9.52 ± 0.75 9.39 ± 0.48 10.00 ± 0.47 10.21 ± 0.70 Phospholipid 34.46 ± 1.66 34.13 ± 1.25 35.25 ± 1.56 34.03 ± 1.09 Ganglioside 0.89 ± 0.07 0.82 ± 0.05 0.93 ± 0.09 1.05 ± 0.06 ───────────────── ─────────────────── Cont: control group Lac: lactose administration group NANA: N-acetylneuraminic acid administration group SL: sialyl lactose administration group The values in the table are averages Values ± standard error, unit is mg / g brain wet weight.

Although there was no significant difference in the amount of other lipids in the whole brain, the ganglioside content was significantly different in the N-acetylneuraminic acid-administered group than in the lactose-administered group (p <0.05).
In the sialyl lactose administration group, the control group, lactose administration group and N
-Significant difference compared to acetylneuraminic acid administration group
(p <0.05). No change was found in the composition of gangliosides in the brain.

[0025]

[Test Example 2] The sialyl lactose obtained in Reference Example 1 was used to examine the effect of long-term administration on the suppression of brain ganglioside content reduction. In addition, 12-month-old SD rats (Charles River Japan) were used as experimental animals.
First, all rats were preliminarily reared on a standard diet (AIN-93G) for 7 days, then divided into two groups each consisting of 30 rats, and each group was administered a feed having the composition shown in Table 3.

[0026]

[Table 3] ────────────────────────── Cont SL ────────────────── ──────── α-corn starch 13.2 13.2 corn starch 39.7 39.7 milk casein 20.0 20.0 white sugar 10.0 6.0 soybean oil 7.0 7.0 crystalline cellulose powder 5.0 5.0 mixed with minerals ¹⁾ 3.5 3.5 mixed with vitamins ²⁾ 1.0 1.0 L-cystine 0.3 0.3 Choline bitartrate 0.25 0.25 Tertiary butyl hydroquinone 0.0014 0.0014 Sialyl lactose-0.2 ────────────────────────── Cont: Control group SL: Sialyl lactose administration Group ¹⁾ : AIN-93-G-MX-OYC ²⁾ : AIN-93-VX-OYC

Rats are bred at a humidity of 60%, a room temperature of 24 ° C., and li.
The ght-dark control was performed for 12 hours, and the rats were fed with food and ion-exchanged water freely. At the start of breeding, and at 3, 6, 9 and 12 months after breeding, 6 rats in each group were randomly anesthetized with ethyl ether to extract the whole brain, and the weight of the brain was measured. After freeze-drying, the ganglioside content in the brain was analyzed. The result is shown in FIG. It is generally said that rats age about 30 times faster than humans, and a 12-month-old rat corresponds to a 30-year-old human. The ganglioside content in the whole brain was significantly different in the sialyl lactose administration group than in the control group (p <0.05).

[0028]

[Test Example 3] Using the sialyl lactose obtained in Reference Example 1, a water maze experiment was conducted in order to examine the effect of differences in whole brain ganglioside content on learning behavior. As an experimental animal, an 8-week-old male SD rat (Charles River Japan) was used. First, all rats were preliminarily reared on a standard diet (AIN-93G) for 7 days, and then divided into 4 groups each consisting of 6 animals, and a feed having the same composition as shown in Table 1 of Test Example 1 was added to each group. Groups were dosed. Raising rats
The test was performed under the conditions of a humidity of 60%, a room temperature of 24 ° C., and a light-dark control of 12 hours.

Then, the "water fille" as shown in FIG.
d multiple T-maze "and the vertical and horizontal length are each 120
Combining a T-shaped maze with an aquarium with a depth of 40 cm and a T-shaped maze, arranging 11 blind paths,
m., vol.27, pp.9-12, 1978). 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
12 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. The required time from the starting point to the target point on the first to third days from the start of the water maze experiment was significantly (p <0.05) shorter in the sialyl lactose-administered group than in the control group and the lactose-administered group. On the second day after the start of the water maze experiment, the time required from the starting point to the target point was significantly greater in the sialyl lactose administration group than in the N-acetylneuraminic acid administration group (p <0.0
5) It was short.

[0030]

BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, sialyl lactose is used as an active ingredient of a brain ganglioside content decrease inhibitor which suppresses a decrease in brain ganglioside content. This sialyl lactose is formulated into a formulation together with a carrier and other conventional components used in the formulation. As for the form of the preparation, it may be orally administered usually as tablets, such as sugar-coated tablets and tablets, granules, solutions, capsules and the like. Further, the sialyl lactose may be used by blending it with a food or drink containing a nutritional composition or the like. Examples of such foods and drinks include yogurt, drinks, cheese, processed milk, and the like. In order to exert the effect of suppressing the decrease in ganglioside content in the brain, it is advisable to ingest at least 10 mg / kg body weight, preferably 30 to 100 mg / kg body weight of sialyl lactose per adult day.

[0031]

Example 1 The sialyl lactose obtained in Reference Example 1 was used.
5 g was filled into a gelatin capsule 000 for internal use of the Japanese Pharmacopoeia to produce a brain ganglioside content decrease inhibitor which suppresses a decrease in brain ganglioside content.

[0032]

Example 2 19.4 kg of warm water was added to 3 kg of skim milk powder, stirred, sterilized at 95 ° C for 10 minutes, and cooled to 42 ° C. Lactobacillus bulgaricus (Lactobaci
llusbulugaricus) and a mixed starter MR of Streptococcus thermophilus
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 71.5 g of sialyl lactose obtained in Reference Example 1 were added to 7.3 kg of water, stirred and dissolved, sterilized at 90 ° C. for 10 minutes, cooled to 10 ° C. Was prepared. Then, the above culture was added to this carbohydrate solution while stirring, and the mixture was uniformly mixed, and then homogenized with a homogenizer to produce 30 liters of drink yogurt. This was filled in a 1-liter paper container to obtain a drink yogurt having an effect of suppressing a decrease in brain ganglioside content. This drink yogurt 1
One liter contains 2 g of sialyl lactose.

[0033]

EFFECTS OF THE INVENTION Sialyl lactose has the effect of suppressing the decrease in ganglioside content in the brain and has the effect of improving brain functions such as improvement of learning behavior. It is useful as a raw material for medicines and foods and drinks that improve and improve brain function.

[Brief description of the drawings]

FIG. 1 shows changes in ganglioside content in the brain with aging of rats in Test Example 2.

FIG. 2 shows a plan view of a T-shaped water maze used in Test Example 3.

[Explanation of symbols]

 1-11 blind road number

FIG. 3 shows the time required for a rat to reach a target point from a starting point of a T-shaped water maze according to Test Example 3.

──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Kyoko Yabe 16-10 Asahicho, Tokorozawa-shi, Saitama 303 Bellami Tokorozawa 303 (72) Inventor Seiichiro Aoe 2-8-9-406, 58-2, Shin-Sayama, Sayama-shi, Saitama Field (Int.Cl. ⁷ , DB name) A61K 31/70 A23L 1/30 C07H 7/027 CA (STN)

Claims (5)

(57) [Claims] 1. A brain ganglioside content decrease inhibitor comprising sialyl lactose or a salt thereof as an active ingredient, which suppresses a decrease in brain ganglioside content. 2. The brain ganglioside content decrease inhibitor according to claim 1, wherein the agent suppresses a decrease in ganglioside content in the brain accompanying the aging of the elderly or the elderly. 3. A food or drink having a brain ganglioside content reduction inhibitory action, characterized by containing sialyl lactose or a salt thereof to suppress a reduction in brain ganglioside content. 4. The method according to claim 1, wherein the sialyl lactose is derived from milk. 5. The inhibitor according to claim 4, wherein the milk-derived sialyl lactose is sialyl lactose derived from any milk selected from the group consisting of cheese whey, bovine colostrum and human milk. Food and drink with action.