JPH07188029A - Antihypertensive agent - Google Patents

Abstract

PURPOSE:To obtain an antihypertensive agent effective for the vasodepression of essential hypertension and capable of being used as a medicine and a food additive. CONSTITUTION:This antihypertensive agent contains as an essential ingredient a RNA obtained from Enterococcus faecalis, Escherichia coli, a yeast, an animal organ, etc., or the treated product of the RNA.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an antihypertensive agent containing ribonucleic acid (RNA) extracted from cells or a treated product thereof as an active ingredient.

[0002]

2. Description of the Related Art Conventionally, various kinds of antihypertensive agents (hypotensive agents) have been used for the prevention or treatment of hypertension. The antihypertensive agents currently in clinical use are antihypertensive diuretics,
There are β-blockers, sympathetic nerve inhibitors, vasodilators, calcium antagonists, etc., but they have various side effects. In the treatment of essential hypertension, secondary diseases caused by side effects of long-term administration of drugs, such as hyperlipidemia, renal disorder due to electrolyte abnormality, sexual dysfunction, and neuropathy, have become a problem.
Moreover, in the treatment of secondary hypertension caused by the presence of a causative disease, the side effects of the above drug administration are more serious. That is, in secondary hypertension, improvement of hypertension is not seen unless the underlying underlying disease is improved, and it is difficult to use antihypertensive agents having side effects.

Since a blood pressure lowering drug is a drug to be administered for a long period of time, there is a demand for the development of a blood pressure lowering drug having no side effect and a strong pharmacological action.

[0004] Recently, microorganism-derived antihypertensive agents have been attracting attention as they have no side effects. Examples of the antihypertensive agent derived from a microorganism include a bacterial cell of Enterococcus faecalis by the present inventor or a treated product thereof (JP-A-5-201871), a glycoprotein having a molecular weight of 10,000 or more isolated from a chlorella algal cell (JP-B-60- 45603), a polysaccharide in a high molecular weight fraction of Lactobacillus (M. Furushiro et al., Agric. Biol. Chem.,
54, 2193-2198 (1990)) and the like are known.

[0005]

The conventional high molecular fraction of microbial cells as an antihypertensive agent with few side effects contains a wide variety of constituents, so that it is difficult to keep the quality constant, and there is a problem in clinical use. It is easy to occur. The present invention provides a superior antihypertensive agent by obtaining a homogeneous antihypertensive agent by fractionating a constituent component having a blood pressure lowering effect from microorganisms, and further treating this.

[0006]

In order to solve this problem, the present inventors separated a polymer fraction extracted from microbial cells into various components by molecular weight fractionation and column adsorption method. When these components are applied to spontaneously hypertensive rats (hereinafter referred to as SHR), which are commonly used as a model animal for human essential hypertension, the RNA fraction and its processed products have a remarkable effect of lowering blood pressure. The present invention has been completed and the present invention has been completed.

That is, the antihypertensive agent provided by the present invention contains the intracellular RNA fraction and its processed product as active ingredients.

As a method for extracting this fraction, the components extracted from the cells with water or hot water may be fractionated by the molecular weight fractionation method or the column adsorption method, or the components may be extracted from the cells using an organic solvent or the like. There are several methods such as direct extraction, but R
Any method may be used as long as it is an operation for fractionating NA.

As shown in the examples, it was confirmed by experiments using SHR that the RNA-containing fraction exhibits a remarkable antihypertensive effect. In addition, RNA is added to this RNA fraction.
The blood pressure-lowering effect was also observed when the treated product treated with the degrading enzyme was used. The effect appeared even when the fragmented RNA was administered. Moreover, not only RNA derived from microorganisms but also Example 1
As shown in FIG. 4, RNA extracted from animal cells also has a blood pressure lowering action, and thus has a blood pressure lowering action regardless of the structure of RNA peculiar to species. Since the RNA-containing fraction was extracted from intestinal indigenous microorganisms or an edible substance, it had almost no toxicity, and no abnormality was observed when SHR was orally or intravenously administered. In addition, there is almost no toxicity whether it is a purified product from a natural product or a synthetic product.

Since the RNA-containing fraction of the present invention and the treated product thereof are water-soluble, they can be easily made into arbitrary dosage forms. The administration method can be either oral administration (including sublingual administration) or parenteral administration (intravenous injection, infusion, etc.). Tablets, granules, powders for oral administration,
In the case of making capsules and the like, generally used binders, inclusion agents, excipients, lubricants, disintegrating agents, wetting agents and the like can be used in the formulation. Also, in the case of a liquid preparation for oral administration, it may be in the form of an aqueous solution for internal use, a shaking agent, a suspension agent, an emulsion, a syrup, etc. by using any auxiliary component.

Although the amount to be used varies depending on the age of symptoms and the like, 0.001 to 0.1 g / kg body weight per day as an active ingredient can be usually administered to an adult once or several times a day.

[0012]

EXAMPLES Examples will be shown below, but the present invention is not limited to the description of these Examples.

Embodiment 1. (Cultivation of Enterococcus faecalis)
s) NF-1011 (Microtechnology Research Institute, No. 12564) was inoculated into Rogosa liquid medium having the composition shown below (number of bacteria: 10 ⁶
Cells / ml), cultured at 37 ° C for 10 to 16 hours, and the viable cell count is about
A culture solution of ⁰⁹ cells / ml was obtained. The obtained culture solution was added to
The cells were collected by centrifugation at 00 × g for 20 minutes and washed twice with distilled water to obtain cells.

The composition of Rogosa liquid medium is shown below. Trypticase 10 g Yeast extract 5 g Tryptoose 3 g Monopotassium phosphate 3 g Dipotassium phosphate 3 g Triammonium citrate 2 g Tween 80 (surfactant) 1 g Glucose 20 g Cysteine hydrochloride 0.2 g Salt solution (as per 1) 5 ml Distilled water 1000 ml (adjusted to pH 7.0, 15 min heat sterilized at 121 ℃) (1) saline; MgSO ₄ · 7H ₂ O 1
_{1. 5g FeSO 4 · 7H 2 O} 0. 68g MnSO 4 · 2H 2 O 2. 4g of distilled water 100ml

Example 2. (Cultivation of Escherichia coli) E. coli K12 strain (ATCC10798) was inoculated into an LB medium having the following composition (the number of bacteria: 10 ⁶ cells / m 2).
l), shaking culture was carried out at 37 ° C. until the viable cell count reached about 10 ⁹ cells / ml. The obtained culture broth was centrifuged at 12,000 × g for 20 minutes to collect the cells, and the cells were washed twice with distilled water to obtain cells.

The composition of the LB medium is shown below. Polypeptone 10 g Yeast extract 5 g NaCl 10 g Glucose 1 g Distilled water 1000 ml (adjusted to pH 7.0, heat sterilized at 121 ° C. for 15 minutes)

Embodiment 3. (Fractionation of Constituent Components from Living Enterococcus) 80 g of the cells obtained in Example 1 was adjusted to pH 7
After suspending in 1 L of a mixed solution of 5 M potassium chloride, 40 mM ammonium acetate and 1 mM magnesium acetate, lysozyme was added to a final concentration of 1,330 μg / ml, and the mixture was kept at 37 ° C. for 2 hours, and then the protoplast cells generated were 4,750 ×
It was collected by centrifugation at 20 g for 20 minutes.

Protoplast cells were treated with 50 mM Tris.
800 ml of HCl solution (pH 8.5), 0.5M EDTA
(PH 8) 80 ml and SLS (sarcosine sodium)
80 ml (Fluka) was added to lyse the cells, which was left overnight at 4 ° C, and then an equal amount of phenol / chloroform (1: 1) was added.
(V / v)) The mixture was mixed and centrifuged at 10,000 × g for 20 minutes to separate an aqueous layer and an organic solvent layer. The recovered aqueous layer was mixed with an equal volume of phenol / chloroform (1: 1 (v
/ V)) Mixing with the mixed solution and separation were repeated twice to extract the nucleic acid fraction. An operation of mixing an equal amount of chloroform with the obtained aqueous layer and removing impurities was repeated twice. Double amount of 100% cold ethanol was added to the obtained aqueous layer to precipitate chromosomal DNA (deoxyribonucleic acid).

After removing the precipitated DNA by winding it with a glass rod, it was cooled at −80 ° C. for 1 hour to precipitate a fraction containing a large amount of RNA, and centrifuged at 4 ° C., 10,000 × g for 20 minutes. To give a precipitate.

The precipitate was treated with 50 mM Tris.HCl (pH
7.0) Dissolve in 1 mM EDTA and 10 mM MgCl ₂ mixed solution, and add DNase I (Takara Shuzo Co., Ltd.) to a final concentration of 50 μ.
It was added to g / ml and reacted at 37 ° C. for 1 hour to remove mixed DNA. After purification with phenol treatment,
Precipitation with ethanol was carried out in the same manner as above to obtain an RNA fraction (referred to as RNA fraction A). RNA fraction A was lyophilized.

Example 4. (Fractionation of Constituent Components from Live Escherichia coli) The live Escherichia coli cells obtained in Example 2 were adjusted to pH 8 with 25% sucrose, 50 mM Tris.HCl and 0.08 M.
The cells were suspended in an EDTA mixture and lysozyme was added to a final concentration of 1 mg / ml to convert the cells into protoplasts. The following operations were carried out according to the method shown in Example 3 to obtain an RNA fraction.

Embodiment 5. (Fractionation of Constituent Components from Heat-treated Enterococcus) The Enterococcus cells obtained in Example 1 had a wet weight of 2
Double the amount of distilled water was added, and heat treatment was performed at 110 ° C. for 10 minutes, followed by centrifugation at 12,000 × g for 20 minutes to collect supernatant fractions and freeze-drying. Supernatant fraction 10g (dry weight)
Was dissolved in 100 ml of distilled water and subjected to ultrafiltration through a membrane having a molecular weight cut off of 10,000 (manufactured by Advantech) to obtain a polymer fraction.

This fraction was fractionated by DEAE-Toyopearl 650 column (2.4 × 13 cm) (manufactured by Tosoh Corporation). FIG. 1 shows the polymer fractionation pattern. After elution and removal of the non-adsorbed fraction (fraction B), the substance eluted by the 0 to 1.4 M sodium chloride concentration gradient elution method was collected in the first half (fraction C) and the second half (fraction D). The molecular weight of each fraction was 3,
It was dialyzed against distilled water with a dialysis membrane of 500 and then freeze-dried.

Example 6. (Measurement of Constituent Content of Extracted Substance) The amount of nucleic acid in the RNA fraction A obtained in Example 3 was measured.
Nucleic acid is a spectrophotometer (U-2000 type manufactured by Hitachi Ltd.)
Was calculated by converting to 1 OD ₂₆₀ = 40 μg / ml RNA at a wavelength of 260 nm. The nucleic acid content of the fraction A was 0.7 mg / ml.

RNA fraction A obtained in Example 3 and enzyme treatment [RNase A (Boehringer Mannheim)
1% agarose gel electrophoresis was performed on the RNA fraction A that had been digested with. The results are shown in Figure 2.

Example 7. (Measurement of Constituent Component Content of Extracted Substance) The amount of nucleic acid was measured for each fraction obtained in Example 5.
The amount of nucleic acid was measured and calculated by the above method. The nucleic acid content of Fractions B and C was 0, and Fraction D was 0.9 mg per mg. Fraction D and enzyme treatment (DNaseI, RNase
Fraction D that had been digested with A) was subjected to 1% agarose gel electrophoresis. The result is shown in FIG. Fraction D
No electrophoretic band appeared in the RNase A-treated product.

Fraction D (20 mg) was placed in an ampoule and 1N-H
₂ ml of ₂ SO ₄ was added, and the tube was sealed under reduced pressure.
The time hydrolysis was performed. Then, the package was opened, neutralized with BaCO ₃ , and BaSO ₄ was removed by centrifugation. This processed product 10μ
l was spotted on a thin layer (Whatman silicagel K5), and ethyl acetate: isopropyl alcohol: water = 65: 22: 1.
Thin layer chromatography was performed with 1 (v / v / v) developing solvent. The results are shown in Fig. 4. Since the treated product of Fraction D showed the same mobility as ribose, Fraction D was identified as an RNA-containing fraction.

Example 8. (Hypotensive effect of oral administration of RNA fraction from live Enterococcus) Male SHR (20) purchased from Charles River Japan
Each group was divided into 8 animals so that the average weight of each group was constant. The RNA fraction A obtained in Example 3 was dissolved in physiological saline, and 65 mg / kg body weight was orally administered by a gastric tube. Physiological saline was administered to the control group.
The systolic blood pressure (mmHg) of the tail artery of a rat kept at 37 ° C for 12 minutes was measured with a non-invasive blood pressure measuring device (MK-1000: manufactured by Muromachi Kikai Co., Ltd.) immediately before administration and 4, 9 and 24 hours after administration. did. The results are shown in Table 1 (measured values represent mean ± SD. The same applies hereinafter).

[0029]

[Table 1]

Example 9. (Hypotensive effect of intravenous administration of RNA fraction from heat-treated Enterococcus) Male SHR (20) purchased from Charles River Japan
Each group was divided into 8 animals so that the average weight of each group was constant.

For the control group, only physiological saline was used, and the various fractions collected in Example 5 were dissolved in physiological saline, respectively.
Each mg / kg body weight was administered into the tail vein. The blood pressure measurement method was the same as in Example 8. Blood pressure was measured immediately before administration and 3 and 24 hours after administration. The results are shown in Table 2. All fractions showed hypotensive effect, especially RNA of fraction D
The contained fraction was remarkable.

[0032]

[Table 2]

Example 10. (Hypotensive effect by intravenous administration of fraction D treated with RNA degrading enzyme) Male SHR (20) purchased from Charles River Japan
Each group was divided into 8 animals so that the average weight of each group was constant.

RNase A (100 mg / ml) was added to the solution obtained by dissolving Fraction D obtained in Example 5 in 10 mg / ml, and the mixture was kept at 37 ° C. for 3 days.
Fraction reacted for time and treated with RNase A (designated as Fraction E)
Was prepared. Fraction E was subjected to an experiment after confirming the disappearance of RNA by 1% agarose gel electrophoresis.

As a control group, 1 RNase A was added to physiological saline.
A solution dissolved in a concentration of 00 mg / ml was used, and 20 mg / kg body weight of the administration sample was administered into the tail vein of each administration group. The blood pressure measurement method was the same as in Example 8. Blood pressure was measured immediately before administration and 3 and 24 hours after administration. The results are shown in Table 3. Fraction D and Fraction E showed almost the same drop rate.

[0036]

[Table 3]

Example 11. (Hypotensive action of oral administration of RNA fraction) Male SHR (20% purchased from Charles River Japan)
Each group was divided into 8 animals so that the average weight of each group was constant.

Each of the fractions D and E prepared by the above method was dissolved in physiological saline, and 65 mg / kg body weight of each was orally administered by a gastric tube (the physiological saline was administered to the control group). ), Blood pressure was measured in the same manner as in Example 8. The results are shown in Table 4 by oral administration of fractions D and E:
A hypotension similar to that of intravenous administration was observed.

[0039]

[Table 4]

Example 12. (Hypotensive effect of oral administration of RNA fraction derived from Escherichia coli) Male SHR (20% purchased from Charles River Japan)
Each group was divided into 8 animals so that the average weight of each group was constant. The Escherichia coli RNA fraction obtained in Example 4 was dissolved in physiological saline, and 65 mg / kg body weight was orally administered by a gastric tube (physiological saline was administered to the control group). Blood pressure was measured. As a result, as shown in Table 5, administration of the RNA fraction derived from Escherichia coli showed a decrease of about 10% in blood pressure value before administration.

[0041]

[Table 5]

Example 13 (Hypotensive effect by oral administration of yeast-derived RNA) Male SHR (20% purchased from Charles River Japan)
Each group was divided into 8 animals so that the average weight of each group was constant.

RNA from Saccharomyces baker's yeast (R6750 Type XI: Purified from Baker)
s yeast) was dissolved in physiological saline and orally administered in the same manner as in Example 11. The results are shown in Table 6
Blood pressure was decreased by administration of A.

[0044]

[Table 6]

Example 14 (Hypotensive effect of oral administration of RNA derived from bovine liver) Male SHR (20% purchased from Charles River Japan)
Each group was divided into 8 animals so that the average weight of each group was constant.

RNA from bovine liver (R72, manufactured by Sigma)
50 Type IV: Purified from Calf Liver) was dissolved in physiological saline and orally administered in the same manner as in Example 11. The results are shown in Table 7. Administration of RNA derived from bovine liver showed about 10% decrease in blood pressure before administration.

[0047]

[Table 7]

Example 15 (Preparation Example) (1) 50 mg of the RNA fraction obtained in Example 3, 4 or 5 or a treated product thereof, 50 mg of purified starch powder and 200 lactose
Mix with mg to give tablets or granules.

(2) 100 mg of the RNA fraction obtained in Example 3, 4 or 5 or a treated product thereof is mixed with 100 mg of soybean protein and 200 mg of lactose to give tablets or granules.

[0050]

EFFECTS OF THE INVENTION The antihypertensive agent of the present invention has almost no side effects and toxicity, and has a remarkable effect on essential hypertension and an immediate effect, regardless of whether it is administered orally or by injection. By having these characteristics, it is possible to prevent hypertension from being added to daily foods as well as being used as a medicine. Further, since RNA may be derived from any source, it is possible to effectively use the discarded ones such as organs (ovary, bone marrow, testis, etc.) of animals and seafood having a high RNA content.

[Brief description of drawings]

FIG. 1 is a diagram showing a fractionation pattern of a heat-treated product of Example 5 by an ion exchanger.

FIG. 2 is a diagram showing agarose gel electrophoresis patterns of Fraction A of Example 6 and an enzyme-treated product thereof.

FIG. 3 is a diagram showing agarose gel electrophoresis patterns of Fraction D of Example 7 and an enzyme-treated product thereof.

FIG. 4 is a diagram showing a thin-layer chromatogram of the Fraction D hydrolyzate of Example 7.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Tetsuro Yamamoto 69, 1406 Entokuin, Ayama-cho, Ayama-gun, Mie Prefecture 69 (72) Inventor Hiroshi Kotsuki 6 Washinton Corporas 203, 1418, Kuwamachi, Ueno-shi, Mie

Claims (5)

[Claims] 1. A hypotensive agent containing ribonucleic acid (RNA) or a processed product thereof as an active ingredient. 2. The antihypertensive agent according to claim 1, wherein the ribonucleic acid is obtained from Enterococcus faecalis cells. 3. The antihypertensive agent according to claim 1, wherein the ribonucleic acid is obtained from Escherichia coli cells. 4. The antihypertensive agent according to claim 1, wherein the ribonucleic acid is obtained from yeast of the genus Saccharomyces. 5. The antihypertensive agent according to claim 1, wherein the ribonucleic acid is obtained from an organ of an animal such as liver.