JP2766439B2 - Cholesterol suppressant - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a cholesterol inhibitor containing β-glucan produced by culturing a microorganism belonging to the genus Macrophomopsis as an active ingredient.

[0002]

2. Description of the Related Art In recent years, fats or cholesterol are frequently consumed, and adult diseases such as hyperlipidemia and arteriosclerosis are increasing. There is a desire for a diet that maintains adequate dietary cholesterol, but there is a tendency to eat increasingly high-fat, high-cholesterol diets. There is a demand for an effective cholesterol elevation inhibitor that keeps the cholesterol level in a living body properly from the cholesterol that is excessively consumed. There is also a need for an effective cholesterol-lowering agent for hypercholesterolemia in which cholesterol levels have already increased.

[0003] Substances having an effect of suppressing cholesterol elevation, such as pectin and guar gum, are known among dietary fibers (Nutrition Journal, Vol. 33, p. 273, 1975), but their effects are weak and they show suppression of cholesterol elevation. Has the disadvantage of requiring large intakes. In addition, a bile salt sequestering agent (bile salt sequence)
Cholestyramine and other resinous substances, which are lanthanides, also show a cholesterol elevation inhibitory effect, but must be taken in large amounts as pharmaceuticals (annual)
Review of Pharmacology; Annual Re
View of Pharmacology, Volume 13, 2
87, 1973), and further has the disadvantage of having side effects that cause nausea and constipation.

[0004]

SUMMARY OF THE INVENTION Accordingly, an object of the present invention is to provide a cholesterol inhibitor which can be used as a pharmaceutical or food material and which shows a strong effect in a small amount.

[0005]

SUMMARY OF THE INVENTION An object of the present invention is to provide a β-glucan produced by culturing a microorganism belonging to the genus Macrophomopus,
-Glucan with a molecular weight of 100,000 to 3,000,000
This is achieved by a cholesterol inhibitor containing a low-viscosity β-glucan whose molecular weight has been reduced to zero.

Hereinafter, the present invention will be described in detail.

[0007] The β-glucan used in the present invention is a filamentous fungus, Macrophomosis.
β-glucan obtained by culturing a microorganism belonging to the genus omopsis) and having the following characteristics. a)
All D-glucopyranosyl residues in the main chain are β-1,3 bonds. b) It has a side chain consisting of one β-1,6 bond D-glucopyranosyl residue at a ratio of one to four D-glucopyranosyl residues in the main chain.

The production of β-glucan has already been studied (Japanese Patent Application Laid-Open No. 2-122701), but its outline is given below.

[0009] Examples of the microorganism used for the production of β-glucan include a microorganism named Macrophomopsis KAB55 strain deposited under Microfabrication Research Commission No. 9366.

Examples of the carbon source used for culturing the filamentous fungus include glucose, maltose, starch, sucrose, fructose, molasses, and mixtures thereof.
When these saccharides are used, β-glucan and α-glucan as a by-product are simultaneously accumulated in the culture, but if a sugar containing galactose as a skeleton as a carbon source is used, microorganisms belonging to the genus Macrophomopsis can be used. In addition, it is possible to selectively produce and accumulate only β-glucan in a culture (Japanese Patent Application No. 3-201002).

Examples of sugars containing galactose as a skeleton include galactose, lactose, melibiose,
Raffinose, stachyose and the like can be mentioned, but lactose is particularly preferred in that the yield of β-glucan is high.

As the nitrogen source, almost all organic and inorganic nitrogen sources used for culturing microorganisms can be used. For example, absorbent cottonseed powder (Pharmamedia), corn steep liquor, yeast extract, dried yeast, Examples include various peptones, oatmeal meat extract, casein hydrolyzate, ammonium salts, nitrates and the like.

Other additives include inorganic salts such as sodium chloride, magnesium, calcium, and phosphoric acid.

The medium may further contain a small amount of a metal salt such as iron, copper or manganese, if necessary.

The culture conditions are preferably pH 3.5 to 9.0, more preferably pH 5.0 to 8.0, and the culture temperature is 1
0 to 40 ° C is preferable, and more preferably 20 to 35 ° C.
And usually cultured for 3 to 7 days.

After completion of the culture, the culture is treated by an appropriate method such as filtration or centrifugation to remove the microorganism. Next, an appropriate precipitant, for example, an organic solvent such as ethanol, methanol, isopropanol, propanol, or acetone is added to the obtained filtrate or supernatant to precipitate β-glucan. The precipitate is separated by a suitable method such as filtration or centrifugation, and then redissolved in water. After repeated precipitation with a precipitant, dialysis and lyophilization yield purified β-glucan. .

The β-glucan thus obtained is highly viscous. However, in any process from the formation of the purified β-glucan and the precipitate from the culture solution to the purification, alkali treatment,
A low-viscosity β-glucan can also be obtained by performing a viscosity reduction treatment such as partial decomposition with an endoglucanase and ultrasonic treatment.

The processing conditions for reducing the viscosity are β
-The molecular weight of the finally obtained β-glucan differs depending on the degree of purification of the glucan, the concentration of β-glucan, etc.
Processing temperature to be 3,000 to 3,000,000,
It is preferable to adjust the processing time and the like. This low-molecular β-glucan has an advantage that it is easy to handle because of its low viscosity.

In the cholesterol inhibitor of the present invention, various additives, excipients, etc., in addition to the β-glucan obtained as described above, can be appropriately blended as long as the object of the present invention is not impaired.

The cholesterol inhibitor of the present invention is administered orally. The dosage varies depending on the age, body weight, symptoms, etc. of the patient. When administered to an adult, generally, the amount of β-glucan per day is 10 to 5,000 mg, more preferably 100 to 1,000 mg. I have.

[0021]

The cholesterol inhibitor of the present invention not only suppresses an increase in the cholesterol level in a living body but also has a cholesterol level-lowering effect effective for hyperlipidemia.

The present invention and its effects will be specifically described below with reference to Production Examples, Test Examples and Examples. Note that the present invention is not limited by this.

Production Example 1 A strain KAB55 belonging to the genus Macrophomopsis (No. 9366, accepted by JASME) was cultured in a medium having the composition shown in Table 1 below.
The culture was pre-cultured for 15 days, and 15 l of the culture was inoculated into a 500 l jar fermenter (manufactured by KYF Engineering Co., Ltd.) containing 300 l of a medium having the same composition, and cultured at 25 ° C. for 4 days. Aeration amount is 1.2 vvm, stirring rotation speed is 60 rpm
The main culture was carried out.

[0024]

[Table 1]

The obtained culture solution was subjected to 8000 revolutions / min.
The cells were removed by centrifugation for 1 minute, and 20% by volume of isopropanol was added to the supernatant to precipitate β-glucan. This is centrifuged at 10,000 rpm for 5 minutes.
β-glucan was recovered. The recovered β-glucan was dissolved in water again, and the above operation was repeated to remove contaminants. After drying, 1.6 g of β-glucan was obtained per 1 liter of the culture solution. Hereinafter, this is referred to as high-viscosity β-glucan.

Production Example 2 20 g of the β-glucan obtained in Production Example 1 was dissolved in 21 of water.
Further, 120 g of NaOH was added and dissolved. After standing at room temperature for 30 minutes, the mixture was neutralized by adding normal hydrochloric acid.
Β-glucan was precipitated by adding 0 ml of isopropanol. This was centrifuged at 10,000 rpm for 5 minutes to recover β-glucan. The recovered β-glucan was dissolved again in water, and the precipitation of β-glucan with isopropanol and the collection by centrifugation were repeated once to remove contaminants, followed by drying to obtain 18 g of β-glucan.

The glucan obtained by the alkali treatment was measured for viscosity at 25 ° C. in a 1% by weight aqueous solution using a bismetholone rotational viscometer. The molecular weight was measured by HPLC (gel filtration column). Table 2 shows the results. The alkali treatment significantly reduced the viscosity of β-glucan and also reduced the molecular weight. Hereinafter, this is referred to as low-viscosity β-glucan.

[0028]

[Table 2]

Test Example 1 (Cholesterol Elevation Suppressing Effect Test) SD male rats (5 weeks old) were preliminarily reared for 4 days on a commercial diet MF (manufactured by Oriental Yeast Co., Ltd.), and then 6 groups per group so that their body weights were almost equal. The animals were divided into five groups. Cholesterol and sodium cholate were added to commercially available feed MF (manufactured by Oriental Yeast Co., Ltd.) so that the final contents became 1% by weight and 0.2% by weight, respectively, to prepare a high cholesterol diet (control feed). In addition, a high cholesterol diet (test diets A to C) in which the high-viscosity β-glucan produced in Production Example 1 was added to the control diet as a test diet, or cholestyramine (a product of Sigma), a known cholesterol inhibitor, was used.
Was added to give a final content of 0.5% by weight to prepare a high cholesterol diet (comparative feed).

The five types of feeds were fed freely to five groups of rats. Immediately before and after transition to high cholesterol diet 5
One day later, blood was collected from the tail vein, and the serum cholesterol level was measured using a commercially available clinical test reagent (Cholesterol E test, manufactured by Wako Pure Chemical Industries, Ltd.).

Table 3 shows the test results. Administration of the high-viscosity β-glucan, which is a cholesterol-lowering agent of the present invention, showed a dose-dependent effect of suppressing a rise in blood cholesterol. The serum cholesterol level of the experimental group to which 0.2% by weight of the high-viscosity β-glucan was administered (test diet C administration group) was significantly lower than that of the experimental group to which the control diet was applied,
A cholesterol elevation inhibitory effect was observed with a small amount of administration (based on Duncan's multiple comparison).

In addition, the serum cholesterol level of the experimental group to which 0.2% by weight of the high-viscosity β-glucan was administered as a diet (the group to which the test feed C was administered) was as follows. Lower than the value of the (administration group), indicating that high-viscosity β-glucan has a cholesterol increase inhibitory effect that is not less inferior to cholestyramine.

[0033]

[Table 3]

The numbers in the table are mean ± standard error. The experimental groups (a, b, c) with different average superscripts were Dunca
There is a significant difference (p <0.05) in the statistical analysis by the n method.

Test Example 2 (Cholesterol lowering test) SD male rats (5 weeks old) were preliminarily reared for 4 days on a commercial diet MF (manufactured by Oriental Yeast Co., Ltd.), and then 7 rats per group so that their body weights were almost equal. Each was divided into two groups. These rats were allowed to freely ingest the high cholesterol diet (control feed) used in Test Example 1 for 3 days to induce hypercholesterolemia.

The two groups of hypercholesterolemic rats 2
Among the groups, one group was used as a test group, and the high cholesterol diet (test feed B) used in Test Example 1 was freely taken for 2 days, and the cholesterol lowering effect of the high-viscosity β-glucan was examined.

The other control experiment group was allowed to freely take a control feed. Immediately before shifting to a high cholesterol diet,
Blood is collected from the tail vein 3 days and 5 days after transfer, and a commercially available clinical test reagent (Cholesterol E test, manufactured by Wako Pure Chemical Industries, Ltd.)
Was used to measure serum cholesterol levels.

Table 4 shows the test results. By administering a high cholesterol diet for 3 days, the serum cholesterol level of both the test group and the control group was significantly increased, and hypercholesterolemia could be induced. The rats in the control group continued on the high cholesterol diet for two more days,
No decrease in serum cholesterol was observed. On the other hand, the rats in the test group were significantly reduced in serum cholesterol level (based on a paired t-test) by administering 1% by weight of a high-viscosity β-glucan diet, and the blood cholesterol level of the high-viscosity β-glucan was reduced. A lowering effect was observed.

[0039]

[Table 4]

The numbers in the table are mean ± standard error. ^*
Statistical analysis by t test showed significant difference from control (p <
0.01), there is a significant difference in the t-test corresponding to the value of the test group on the 3rd day after the transition to the high cholesterol diet (p <0.00).
1).

Test Example 3 (Cholesterol Suppression Test) SD male rats (5 weeks old) were preliminarily reared for 4 days on a commercial diet MF (manufactured by Oriental Yeast Co., Ltd.), and then 6 rats per group so that their body weights were almost equal. They were divided into three groups.

The high-viscosity β-glucan produced in Production Example 1 and the low-viscosity β-glucan produced in Production Example 2 were added to the control feed used in Test Example 1 so that the final content was 1% by weight. A cholesterol diet (test feeds D and E) was prepared. These two types of test feed and control feed were each allowed to freely ingest three groups of rats. Blood is collected from the tail vein immediately before and 5 days after the transfer to a high cholesterol diet, and a commercially available clinical test reagent (Cholesterol E test, manufactured by Wako Pure Chemical Industries)
Was used to measure serum cholesterol levels.

Table 5 shows the test results. The serum cholesterol level 5 days after the shift to the high cholesterol diet was significantly lower in both the test feed D administration group and the test feed E administration group than in the control feed administration group. In addition, test feed D
The serum cholesterol values of the administration group and the test feed E administration group are almost equal, and the β-glucan that has been made less viscous by the alkali treatment, like the untreated high-viscosity β-glucan,
It was shown to have a cholesterol-suppressing effect.

[0044]

[Table 5]

The numbers in the table are mean ± standard error. The experimental groups (a, b) having different average superscripts have a significant difference (p <0.05) in the statistical analysis by the Duncan method.

Test Example 4 (Safety Test) SD male rats (5 weeks old) were preliminarily reared for 4 days on a commercial diet MF (manufactured by Oriental Yeast Co., Ltd.), and then 6 rats per group so that their body weights were almost equal. They were divided into two groups. The test feed A or the commercial feed MF (manufactured by Oriental Yeast) used in Test Example 1 was administered to the test group and the control group, respectively, and observed for 7 days.

The dose of β-glucan to rats estimated from feed intake was 5 g / kg or more per day.
During this period, no rats died and no abnormalities were observed in behavioral observation. In addition, no significant difference was observed in the change in weight gain from the control group.

Example 1 [Tablets] The highly viscous β-produced in Preparation Example 1 as an active ingredient in one tablet
Tablets containing 100 mg of glucan were prepared as follows.

[0049]

[Table 6]

(Preparation method) Hydroxypropyl cellulose was dissolved in 30 g of water and added to a mixture of high-viscosity β-glucan, lactose, corn starch and crystalline cellulose, and kneaded well. The kneaded product was passed through a 20-mesh sieve, granulated into granules, and dried. After that, the obtained granules were mixed with magnesium stearate and tableted into 200 mg per tablet.

Example 2 [Capsules] Capsules containing 100 mg of the highly viscous β-glucan produced in Production Example 1 as an active ingredient in one tablet were prepared as follows.

[0052]

[Table 7]

(Preparation method) The above components were sufficiently mixed, and 300 mg of the mixture was filled in No. 2 capsules to obtain capsules.

Example 3 [Granules] Highly viscous β-produced in Production Example 1 as an active ingredient in 1 g
A granule containing 100 mg of glucan was prepared as follows.

[0055]

[Table 8]

(Preparation method) Hydroxypropylcellulose dissolved in 30 g of water was added to a mixture of high-viscosity β-glucan, lactose and corn starch, and kneaded well. The kneaded product was passed through a 20-mesh sieve, granulated, dried, and sized to obtain granules.

Example 4 [Drink] A granule containing 300 mg of the highly viscous β-glucan produced in Production Example 1 as an active ingredient in 100 ml was prepared as follows.

[0058]

[Table 9]

(Preparation method) The above components (a) to (d) were added and dissolved in 850 ml of purified water at 60 ° C., and then the above component (e) was added and mixed well. Add purified water to this and add 1
After further mixing well to make 000 ml, the mixture was heated to 90 ° C. and hot-packed into drink bottles in 100 ml portions to obtain a drink.

[0060]

As described above, the cholesterol inhibitor of the present invention suppresses an increase in the cholesterol level in a living body and is effective for the prevention of hyperlipidemia. Obviously, it also has the effect of lowering the value.

──────────────────────────────────────────────────続 き Continuation of the front page (56) References JP-A-3-127723 (JP, A) JP-A-3-167109 (JP, A) JP-A-60-188402 (JP, A) JP-A-55- 76817 (JP, A) JP-A-4-505997 (JP, A) JP-A-59-175436 (JP, A) (58) Fields investigated (Int. Cl. ⁶ , DB name) A61K 31/715 ADN C08B 37/00

Claims (2)

(57) [Claims] 1. The method of claim 1, wherein the macrophomopsis is
A cholesterol inhibitor containing β-glucan having the following binding mode, which is produced by culturing a microorganism belonging to the genus mopsi). a) All D-glucopyranosyl residues in the main chain are β-1,
There are three bonds. b) 1 D-glucopyranosyl residue having β-1,6 bond at a ratio of 1 to 4 D-glucopyranosyl residues in the main chain
It has side chains consisting of individual pieces. 2. Macrophomopsis (Macrophopsis)
(Mopsis) containing a low-viscosity β-glucan produced by culturing a microorganism belonging to the genus mopsis and having the following binding mode and having a molecular weight of 100,000 to 3,000,000. Cholesterol inhibitors. a) All D-glucopyranosyl residues in the main chain are β-1,
There are three bonds. b) 1 D-glucopyranosyl residue having β-1,6 bond at a ratio of 1 to 4 D-glucopyranosyl residues in the main chain
It has side chains consisting of individual pieces.