JPH0656681A - Improver of blood triglyceride value - Google Patents

Abstract

PURPOSE:To provide the subject improver which contains the cell bodies of a microorganismic strain belonging to the genus Bifidobacterium, and is useful for prevention and therapy of hyperlipemia because it can lower the value of blood triglycerides extremely effectively and safely. CONSTITUTION:The objective improver contains the cell bodies of a strain belonging to the genus Bifidobacterium such as Bifidobacterium bifidum. The cell bodies may be viable or dead, but the viable cells are administered orally in a dose of 1 to 10g (10<8> to 10<11> cells/g of the preparation), 3 times a day. The dead cells also may be administered orally or parenterally, but in the case of subcutaneous injection, it is administered daily 0.1 to 100mg as the suspension. The dead cells are obtained by heating the viable cells.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a blood lipid improving agent.

[0002]

BACKGROUND OF THE INVENTION Blood lipids have important health implications, for example VLDL in plasma in patients with arterial disease.
It is known that one or both of (mainly triglyceride) and LDL (mainly cholesterol) have a high concentration. Conventionally, compounds such as clofibrate and nicotinic acid have been used for the purpose of preventing the synthesis of triglyceride in the body, but they are not sufficiently satisfactory in terms of effects and side effects.

[0003]

DISCLOSURE OF THE INVENTION The present invention is intended to provide a triglyceride value improving agent which is more effective and highly safe.

[0004]

The present invention is a blood triglyceride level improving agent containing cells of a strain belonging to the genus Bifidobacterium.

Examples of Bifidobacterium include, for example, Bifidobacteria Bifidum.
bacterium bifidum), the same as Address Sentis (B. adolescentis), the same
B. breve, B. infantis, and B. lon
gum) and the like.

The cells may be live cells or dead cells and may be administered orally or parenterally, but it is desirable to administer a suspension of dead cells parenterally, for example by subcutaneous injection. .

Viable cells can be easily obtained by culturing Bifidobacterium by a known method. The dead cells can be obtained by sterilizing live cells by heating or freeze crushing. Desirable conditions for heat sterilization are, for example, about 120 ° C. for 10 minutes, but heating at a lower temperature for a long time or at a higher temperature for a short time may be performed.

The bacterial cells are usually used as a preparation diluted with a harmless carrier or solvent. For example, to dilute viable cells,
Carriers such as starch, dextrin, and lactose that do not affect the survival of bacteria are preferably used, and dead cells may be suspended in physiological saline, for example. It is desirable to administer live cells orally, and the preferred dose is 10 ⁴ to 1
0 ¹² , preferably 10 ⁸ to 10 ¹¹ / g of the formulation,
It is 1 to 10 g three times a day.

The dead cells are administered orally or parenterally,
The dosage in the case of oral administration is similar to that in the case of viable cells, but in the case of parenteral administration such as subcutaneous injection, 0.1-1 and
000 mg, preferably 0.1 to 100 mg (1 g corresponds to about 10 ^{12 in} viable cell count) is used.

As is clear from Reference Example 1, Bifidobacterium does not actually show toxic effects on human living cells.

[0011]

In the present invention, the cells of Bifidobacterium suppress the synthesis of triglyceride in the body and reduce the triglyceride level in blood.

Example 1 Preparation of live and dead bacterial cell samples. Bifidobacterium bacteria in the medium of the following composition,
It was cultured under anaerobic conditions at 37 ° C. for 18 hours.

Medium composition (Brigg's Liver broth): Tomato juice leachate 400 ml Neopeptone (manufactured by Difco) 15 g Yeast extract (manufactured by Difco) 6 g Liver extract ^* 75 ml Glucose 20 g Soluble starch 0.5 g Sodium chloride 5 g Tween 80 1 g L-cystine / HCl / H ₂ O 0.2 g Purified water 525 ml pH 6.8

* Liver extract is the end of liver (Kyokuto) 10
After leaching 170 g of purified water in a warm bath at 50-60 ° C for about 1 hour, the mixture was heated at 100 ° C for several minutes to reach pH 7.2.
It was prepared by filtering with a filter paper after being modified to.

After the completion of the culture, the culture solution was centrifuged at 3,000 rpm for 10 minutes to collect the cells, which were washed several times with physiological saline and then suspended in physiological saline or a 0.5% sodium glutamate aqueous solution. It was used as a viable cell sample.

Further, the above-mentioned viable cell sample was subjected to 10 ° C. at 10 ° C.
After heating for a minute, it was freeze-dried and powdered to give a killed bacterial cell sample.

Reference Example 1 Action of Bifidobacterium on human peripheral mononuclear cells. Mononuclear cells separated from heparinized venous blood by Fico-Hypaque gravity centrifugation were added with defatted fetal calf serum 5% and non-essential amino acid (× 100, GIBCO) 0.1% RP.
About 1 × 10 ⁶ / m in MI1640 medium (manufactured by Nissui)
It was floated to a concentration of 1. 1m of this cell suspension
1 and B. bifidum G9-
The killed cell sample of No. 1 was placed in a test tube and cultured in a 5% CO ₂ incubator for 48 hours. 2 hours before the end of culture,
Microcurie ¹⁴ C-acetic acid (New England, New England Company) was added.

At the end of culturing, the culture solution was cooled with cold PBS (Dulb).
ecco phosphate buffered saline) 1 ml and wash with PBS
After suspending in 1 ml, 1 ml of 20% KOH ethanol solution was added and stirred, and heated at 75 ° C. for 1 hour. Immediately after completion of the reaction, 2 ml of hexane was added under ice-cooling for extraction, and 1 ml of a 1% dichitonin solution was added thereto to precipitate the sterol portion. The precipitate thus obtained was collected with a glass filter, the radioactivity in the precipitate was measured with a liquid scintillation counter, and the amount of cholesterol biosynthesis was expressed as dmp (decay per minute). Also,
The trypan blue dye excretion test was used to measure the number of living mononuclear cells in the culture system. The results are shown in Table 1.

[0019]

[Table 1] Radioactive material decay rate of Bifidobacterium Number of living cells of mononuclear cells Concentration of dead cells ^* (dpm) / 1 ml ─────────────────────── ────── Counter 614 ± 10 68 × 10 ⁴ 100 μg / ml 89 ± 1 62 × 10 ⁴ 75 135 ± 15 60 × 10 ⁴ 50 236 ± 18 70 × 10 ⁴ 25 481 ± 12 68 × 10 ⁴ 10 513 ± 36 61 × 10 ⁴ 5 561 ± 25 65 × 10 ⁴ * Approximately 1 × 10 ¹² / g in terms of viable cell count

As is clear from Table 1, the viable cell count of mononuclear cells was almost the same as that in the case of no addition, in the concentration range of killed Bifidobacterium cells of 5 to 100 μg / ml. Killed cells of the genus Umbu have little toxicity to human mononuclear cells and are excellent in safety.

Example 2 Blood triglyceride level lowering action of Bifidobacterium. B. obtained by the method of Example 1 bifidum G9
-1 of a viable cell sample (the number of bacteria: about 3 × 10 ¹⁰ / ml) was applied to 6-week-old rat males (Jcl: 7 SD) at 1.0 ml / animal /
It was administered orally by gavage in a daily dose. The control group (7 animals) was similarly administered with 1.0 ml / animal / day of physiological saline or 0.5 M sodium glutamate.

As the feed, a high cholesterol diet prepared by mixing 1% cholesterol, 0.25% sodium cholate and 5% olive oil with CL-2 powdered feed (manufactured by CLEA Japan, Inc.) was fed at a rate of 15 g per rat per day. As for water, tap water was freely available. After breeding for 2 weeks, blood was collected from the abdominal aorta with a heparin-containing vacuum blood collection tube under anesthesia with Nembuntal (manufactured by Abbott Laboratories), and 3,000 at 5 ° C.
After centrifugation at rpm for 10 minutes, the lipid component in the obtained plasma was measured using the following commercial kit.

Total cholesterol: Cholesterol CII
-Test Wako Free cholesterol: Free cholesterol C-Test Wako Triglyceride: Triglyceride CII-Test Wako (all manufactured by Wako Pure Chemical Industries) The results are shown in Table 2.

[0024]

[Table 2]

[0025] 2) Approximately 3 × 10 ¹⁰ (viable cells) / day administered per rat * P <0.05 significant difference ** P <0.0025 significant difference

As shown in Table 2, the triglyceride level in plasma of the Bifidobacterium-administered group was lower in the Bifidobacterium-administered group than in the control group, and there was a tendency for improvement.

Furthermore, although the occurrence of fatty liver and the increase in liver weight were observed by feeding a high-cholesterol diet, the degree of fatty liver was lighter in the bacterial administration group than in the control group, and the ratio of liver weight to rat body weight was also higher. It turned out to be kept low.

Example 3 Blood triglyceride level lowering action of Bifidobacterium. B. obtained by the method of Example 1 bifidum G9
-1 dry cells (bacteria number about 3 × 10 ¹¹ / g) were mixed with a commercially available feed (CE-2: CLEA Japan, Inc.) at a ratio of 10%, and 1
Four-week-old obese animal Wistar fatty rat
(6 females) were fed and fed. The animals in the control group were fed with only CE-2 as a feed. Feed and water (tap water) were fed ad libitum. After 3 weeks, decapitate and collect whole blood,
The lipid component in plasma was measured in the same manner as in Example 2. The results are shown in Table 3.

[0029]

[Table 3]

The results in Table 3 show that administration of Bifidobacterium can significantly lower the blood triglyceride level.

[0031]

INDUSTRIAL APPLICABILITY According to the improving agent of the present invention, the triglyceride level in blood can be lowered safely and extremely effectively, and an excellent effect for the prevention and treatment of hyperlipidemia can be obtained.

Front page continuation (72) Inventor Koji Shikata 1-7-1, Imafukunishi, Joto-ku, Osaka (72) Mamoru Hatano 5-2-6 Mikage-honcho, Higashinada-ku, Kobe-shi, Hyogo

Claims (6)

[Claims] 1. A blood triglyceride level improving agent comprising cells of a strain belonging to the genus Bifidobacterium. 2. The improving agent according to claim 1, wherein the microbial cell is a live microbial cell. 3. The improving agent according to claim 1, wherein the cells are dead cells. 4. The strain is selected from Bifidobacterium bifidum, the same addressacetis, the same breve, the same infantis and the same longum.
The described improver. 5. The bacterium is a viable bacterium, and the number of bacteria is 10 ⁴ to 1
The blood lipid-improving agent according to claim 1, which is prepared to have a concentration of 0 ¹² , preferably 10 ⁸ to 10 ¹¹ / g. 6. The cells are dead cells, and 0.1 to 1,
The blood lipid improving agent according to claim 1, which is prepared so that 000 mg, preferably 0.1 to 100 mg, can be administered at one time.