JPH058173B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to an antihypertensive agent containing a high-molecular-weight polysaccharide substance MPS-80 as an active ingredient. In general, polysaccharides, which are one of the constituents of food, have been shown to cause colorectal cancer and cancer, based on the results of many epidemiological studies in recent years.
It has become clear that they play an important role in preventing the onset of many diseases such as arteriosclerotic diseases. In particular, as it became clear that certain polysaccharides have the effect of suppressing the rise in blood cholesterol, they have attracted attention from the perspective of prevention and treatment of arteriosclerotic diseases. First, a new polymeric polysaccharide substance was extracted from various lactic acid bacteria.
I came across MPS-80 (patent application 1983-3039),
Since MPS-80 is an excellent polymeric polysaccharide, we conducted extensive research to determine whether it has an effect of suppressing blood pressure rise.We found that MPS-80 has a significant effect of suppressing blood pressure rise, which led us to complete the present invention. . In the present invention, if MPS-80 is continuously administered as an orally administered agent, it is effective in treating and preventing arteriosclerotic diseases and hypertensive diseases. The dosage is approximately MPS-800.1 to 10g/Kg/day. Next, the MPS-80 will be explained. MPS−80 is Lactobacillus jugurti No.851,
FERM BP−66 or Streptococcus
thermophilus No. 127, produced by FERM BP-65. The physical and chemical properties of MPS-80 are as follows. (1) Elemental analysis C: 42.2% H: 6.9% O: 50.4% (2) Molecular weight (i) By ultrafiltration method. The results of ultraviolet analysis using Sepharose 2B are shown in Figure 1. Column size 2.5×40.5cm; fraction 5g;
Loading 2.5 mg (1 ml) of sample; 0.05 M phosphate buffer solution (PH6.0) as developing agent;
It is fractionated near Void Volume. (ii) By ultracentrifugation. A sample dissolved in 0.2M phosphate buffer (PH7.3) at a concentration of 0.1% was subjected to ultracentrifugation (set rotation speed).
51200RPM) to determine the sedimentation constant.
It was 7.98S (S: Svedberg unit). (Sedimentation showed a single state.) (3) Melting point (decomposition point) This substance begins to change color at around 262℃, and reaches 263-264℃.
It turns black at ℃. (4) Specific rotation [a] ¹⁸ _D = +33.2 (C = 0.5%) (5) Ultraviolet absorption spectrum As shown in Figure 2. (6) Infrared absorption spectrum As shown in Figure 3. (7) Solubility in solvents Soluble in water, insoluble in methanol, ethanol, acetone, and ether. (8) Color reaction (i) Moritsch reaction + (ii) Anthrone reaction + (iii) Cysteine-sulfuric acid reaction + (iv) Aniline-hydrochloric acid reaction − (v) Carbazole-sulfuric acid reaction − (vi) Elson-Morgan reaction − (vii) Billetz reaction - (9) Basic, acidic, neutral The pH of a 0.1% to 0.5% aqueous solution of this substance is neutral. (10) Color of substance The lyophilized product of this substance is white fibrous. (11) Types of constituent sugars The types of constituent sugars were investigated by GLC using 5% SE-52 (2m column). Conditions: Temperature increase from 150℃ to 230℃ (3℃/min)
It was hydrolyzed in boiling water with 2N-H ₂ SO ₄ for 4 hours, neutralized with barium carbonate, and filtered. After desalting the liquid with Amberlite IRA-410 and Amberlite IR-120B, it was concentrated to dryness.
It was converted into TMS and subjected to GLC. As a result, glucose and galactose were recognized as constituent sugars of this substance. (12) Composition ratio of constituent sugars A sample was hydrolyzed in boiling water with 2N-H ₂ SO ₄ for 4 hours, neutralized with barium carbonate, filtered, and the composition ratio of constituent sugars was investigated using the enzyme method. As a result, glucose:galactose=2.2-1.9:1
It was hot. (13) C ¹³ -NMR spectrum (in D ₂ O, TMS standard) (ppm) The results are shown in FIG. (14) Degradability by enzymes Various enzymes were allowed to act on this substance dissolved in 0.05M acetate buffer. Enzymatic degradability was determined by the increase in reducing sugar amount using the Somogyi-Nelson method. Enzymes and reaction conditions used. a α-Amylase (Boehringer) PH5.9, 37
°C, 4 hoursb β-Amylase (Boehringer) PH4.8, 30
°C, 4 hours c β-Galactosidase (Boehringer) PH4.8,
30℃, 4 hours d Amyloglucosidase (Boehringer) PH4.8,
30℃, 4 hours e α-Galactosidase (Boehringer) PH4.8,
30°C, 4 hours Under the above conditions, no increase in reducing sugar content was observed in all cases a to e. (15) LD ₅₀ ddY5w♀ mice (average weight 21.3g, 7 mice per group)
Using this method, various doses of samples dissolved in physiological saline were intraperitoneally administered once, observed for 10 days, and the LD ₅₀ was determined. As a result, the LD ₅₀ was 200 mg/Kg body weight or more. Next, production examples and examples of the present invention will be shown. Production example Whey medium (10% w/v whey powder + 0.5% w/
v beer yeast extract) Lactobacillus in 10
Jugurti No.851, inoculated with FERM BP−66, 37℃
After culture is completed, culture supernatant 9.2 is obtained by centrifugation (10,000 rpm, 15 min). To this supernatant, 99.5% ethyl alcohol is added to a final concentration of 35% (v/v). As a result of this operation, precipitated substances can be observed, and these precipitated substances are centrifuged (10,000 rpm,
5 min) to obtain 1.2 g of precipitated material. After dissolving the precipitated material by adding ion-exchanged water, insoluble materials are removed by centrifugation (10,000 rpm, 15 min). By repeating the operation of collecting precipitated substances by adding ethyl alcohol and washing with water three times in total.
800 mg of crude product is obtained. The crude product obtained here is dissolved in 0.05M phosphate buffer (PH6.0) and loaded onto a column packed with diethylaminoethylcellulose (DEAE-cellulose) sufficiently buffered with the same buffer. The same buffer solution is passed through, and the flow-through solution containing substances not adsorbed to DEAE-cellulose is collected. After the solution containing the non-adsorbable substance is freeze-dried, it is dissolved in ion-exchanged water and dialyzed against ion-exchanged water at 10° C. for 4 days in a dialysis tube. After dialysis, the dialysis fluid is freeze-dried and a freeze-dried specimen of the polymeric polysaccharide substance MPS-80 is prepared.
Get 500mg. Example Crude material obtained by alcohol precipitation of the culture solution obtained in the production example (3% MPS-80)
containing) in commercially available rat and mouse breeding feed.
10% was added and mixed. This is used as feed. As a control, 10% whey protein was added to the same commercially available rat and mouse breeding feed to match the protein content and energy levels of the feed. This is used as feed. As animals, spontaneously hypertensive rats (SHR) weighing 40 to 50 g immediately after weaning.
Males were used. This SHR has the property that when raised on general breeding feed, its blood pressure increases significantly compared to normal rats. In the experimental area, 30 SHRs were raised on feed.
In the same experimental area, 30 SHRs were fed with feed. In both experimental groups, blood pressure was measured weekly from changes in the rat's tail artery. The experimental results are shown in Table 1,
FIG. 5 shows this in a graph. As is clear from Table 1 and FIG. 5, blood pressure in the experimental group was consistently lower than in the experimental group after the start of feeding.

【table】

【table】 [Brief explanation of the drawing]

FIG. 1 is a development diagram of the polymeric polysaccharide substance MPS-80 obtained by ultrafiltration. a...High molecular weight polysaccharide substance
MPS-80, b...Blue Dextran Figure 2 shows the ultraviolet absorption spectrum of the polymeric polysaccharide substance MPS-80, Figure 3 shows the same infrared absorption spectrum, and Figure 4 shows the same C ¹³ -NMR spectrum. FIG. FIG. 5 is a diagram showing a comparative test of blood pressure increase using spontaneously hypertensive rats in Examples.

Claims (1)

[Claims] 1. A polymeric polysaccharide substance having the following physical and chemical properties:
A blood pressure increase suppressant containing MPS-80 as an active ingredient. (1) Elemental molecules C: 42.2% H: 6.9% O: 50.4% (2) Molecular weight (i) When using ultrafiltration method. The results of ultrafiltration using Sepharose 2B are shown in FIG. Column size 2.5×40.5cm; fraction 5g;
Loading 2.5 mg (1 ml) of sample; 0.05 M phosphate buffer solution (PH6.0) as developing agent;
It is fractionated near Void Volume. (ii) By ultracentrifugation. A sample dissolved in 0.2M phosphate buffer (PH7.3) at a concentration of 0.1% was subjected to ultracentrifugation (set rotation speed).
51200RPM) to determine the sedimentation constant.
It was 7.98S (S: Svedberg unit). (Sedimentation showed a single state.) (3) Melting point (decomposition point) This substance begins to change color at around 262℃, and reaches 263-264℃.
It turns black at ℃. (4) Specific rotation [a] ¹⁸ _D = +33.2 (C = 0.5%) (5) Ultraviolet absorption spectrum As shown in Figure 2. (6) Infrared absorption spectrum As shown in Figure 3. (7) Solubility in solvents Soluble in water, insoluble in methanol, ethanol, acetone, and ether. (8) Color reaction (i) Moritsch reaction + (ii) Anthrone reaction + (iii) Cysteine-sulfuric acid reaction + (iv) Aniline-hydrochloric acid reaction − (v) Carbazole-sulfuric acid reaction − (vi) Elson-Morgan reaction − (vii) Billetz reaction - (9) Basic, acidic, neutral The pH of a 0.1% to 0.5% aqueous solution of this substance is neutral. (10) Color of substance The lyophilized product of this substance is white fibrous. (11) Types of constituent sugars The types of constituent sugars were investigated by GLC using 5% SE-52 (2m column). Conditions: Temperature increase from 150℃ to 230℃ (3℃/min)
The mixture was hydrolyzed in boiling water with 2N-H ₂ SO ₄ for 4 hours, neutralized with barium carbonate, and then filtered. After desalting the filtrate with Amberlite IRA-410 and Amberlite IR-120B, it was concentrated to dryness.
It was converted into TMS and subjected to GLC. As a result, glucose and galactose were recognized as constituent sugars of this substance. (12) Compositional ratio of constituent sugars A sample was hydrolyzed in boiling water with 2N-H ₂ SO ₄ for 4 hours, neutralized with barium carbonate and filtered, and the filtrate was examined for the compositional ratio of constituent sugars by an enzymatic method. As a result, glucose: galactose = 2.2-1.9:
It was 1. (13) C ¹³ -NMR spectrum (in D ₂ O, TMS standard) (ppm) The results are shown in FIG. (14) Degradability by enzymes Regarding this substance dissolved in 0.05M acetate buffer,
Various enzymes were applied. Enzymatic degradability was determined by the increase in reducing sugar amount using the Somogyi-Nelson method. Enzymes and reaction conditions used. a α-Amylase (Boehringer) PH5.9, 37
°C, 4 hoursb β-Amylase (Boehringer) PH4.8, 30
°C, 4 hours c β-Galactosidase (Boehringer) PH4.8,
30℃, 4 hours d Amyloglucosidase (Boehringer) PH4.8,
30℃, 4 hours e α-Galactosidase (Boehringer) PH4.8,
30°C, 4 hours Under the above conditions, no increase in reducing sugar content was observed in all cases a to e. (15) LD ₅₀ ddY5w♀ mice (average weight 21.3g, 7 mice per group)
Using this method, various doses of samples dissolved in physiological saline were intraperitoneally administered once, observed for 10 days, and the LD ₅₀ was determined. As a result, the LD ₅₀ was 200 mg/Kg body weight or more.