JP2889491B2 - Antihypertensive - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a hypotensive agent containing deoxyribonucleic acid (DNA) extracted from cells as an active ingredient.

[0002]

2. Description of the Related Art Conventionally, various types of antihypertensive agents (hypertensive agents) have been used for preventing or treating hypertension. Antihypertensives currently in clinical use include antihypertensive diuretics,
Although there are many types such as β-blockers, sympathomimetic agents, vasodilators, calcium antagonists, etc., each drug has a wide variety of side effects. In the treatment of essential hypertension, hyperlipidemia, renal disorder due to electrolyte abnormality, sexual dysfunction,
Secondary diseases such as neuropathy due to side effects of long-term administration of drugs have become a problem. Further, in the treatment of secondary hypertension caused by the presence of a causative disease, the above-mentioned side effects due to the administration of the drug are serious. That is, secondary hypertension does not improve hypertension unless the underlying underlying disease is improved, and it is difficult to use antihypertensive drugs that have side effects.

[0003] Antihypertensive drugs are drugs that are administered for a long time. Therefore, there is a demand for the development of a potent antihypertensive agent having no pharmacological action without side effects.

Recently, microorganism-derived antihypertensive agents without side effects have attracted attention. Examples of the microorganism-derived antihypertensive agent include cells of Enterococcus faecalis or a processed product thereof (JP-A-5-201871) by the present inventors.
Glycoproteins having a molecular weight of 10,000 or more isolated from Chlorella algae (Japanese Patent Publication No. 60-45603) and polysaccharides in the high molecular fraction of Lactobacillus (M. Furushiro et al., Agric. Biol. Chem.,
54, 2193-2198 (1990)).

[0005]

The high molecular weight fraction of conventional microbial cells as a hypotensive agent with few side effects contains a wide variety of components, making it difficult to maintain a constant quality, and poses a problem in clinical use. Easy to occur. The present invention
The present invention is intended to obtain a homogeneous antihypertensive agent by fractionating components having a blood pressure lowering action from microorganisms, and to provide a more excellent antihypertensive agent by treating the same.

[0006]

Means for Solving the Problems In order to solve this problem, the present inventors have constructed a polymer fraction extracted from microbial cells by various methods using known fractionation methods such as molecular weight fractionation and column adsorption. Fractionated into components. When these components were applied to a spontaneously hypertensive rat (hereinafter referred to as SHR), which is widely used as a model animal for human essential hypertension, DN
It has been found that the fraction A has a significant effect of lowering blood pressure, and the present invention has been completed.

[0007] That is, the antihypertensive agent provided by the present invention comprises an intracellular DNA fraction as an active ingredient.

The main method of extracting this fraction is to destroy the cells, remove the protein with an organic solvent, and collect the relevant fraction by centrifugation or take-up.
Any method can be used as long as it is an operation for fractionating.

As shown in the examples, the fraction containing DNA showed a remarkable blood pressure lowering effect in an experiment using SHR, and it was confirmed that the lowering effect was persistent. In addition, as shown in Examples,
Not only A but also DNA derived from animal cells has a blood pressure-lowering effect, and thus has a blood pressure-lowering effect irrespective of the structure of the species-specific DNA. Since the fraction containing DNA was extracted from intestinal microorganisms or edible substances, there was almost no toxicity, and no abnormalities were observed when administered orally or intravenously to SHR. Also,
There is almost no toxicity whether it is purified from natural products or synthetic products.

[0010] Large intake of foods containing a large amount of nucleic acids (DNA and RNA) is considered to be one of the causes of hyperuricemia. However, according to recent research, it has been proven that when a food-derived nucleic acid component enters the body, a feedback mechanism acts on the synthesis in the body, and the amount of nucleic acid in the body is kept constant. Therefore, apart from a patient with hereditary gout or a patient with hyperuricemia caused by abnormal purine metabolism due to chromosomal abnormality, no side effect is caused by using the agent of the present invention.

Since the fraction containing the DNA of the present invention is water-soluble, it can be easily made into any dosage form. In addition, the administration method can be either oral administration (including sublingual administration) and parenteral administration (intravenous injection, infusion, etc.).
In the case of tablets, granules, powders, capsules and the like for oral administration, a binder, an emulsifier, an excipient, a lubricant, a disintegrant, a wetting agent, etc., which are generally used in the formulation, are used. It can be used as needed. Also, in the case of a liquid preparation for oral administration, it can be in the form of an aqueous solution for internal use, a shaking mixture, a suspension, an emulsion, a syrup, etc., using any auxiliary component.

The amount used depends on the age of the symptoms and the like, but 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.

[0013]

EXAMPLES The present invention will be described below with reference to Examples, but the present invention is not limited by these Examples.

Embodiment 1 FIG. (Culture of Enterococcus) Enterococcus faecali
s) Inoculation of NF-1011 (Microtechnical Laboratories No. 12564) into Rogosa's liquid medium having the following composition (the number of bacteria: 10 ⁶⁾
Cells / ml) at 37 ° C. for 10 to 16 hours to obtain a culture solution with about 10 ⁹ viable cells / ml. The obtained culture solution is
The cells were collected by centrifugation at 2,000 × g for 20 minutes and washed twice with distilled water to obtain cells.

The composition of Rogosa liquid medium is shown. Trypticase 10 g Yeast extract 5 g Tryptose 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, 121 ° C. 15 minutes heat-sterilized in) (1) _{saline: MgSO 4 · 7H 2 O 11.5g} FeSO 4 · 7H 2 O 0.68g MnSO 4 · 2H 2 O 2.4g distilled 100 ml of water

Embodiment 2 FIG. (DNA extraction from live cells) 80 g of the cells obtained in Example 1 were adjusted to pH7.
5 M potassium chloride, 40 mM ammonium acetate and 1
Suspended in 1 L of a mixed solution of mM magnesium acetate, lysozyme was added to a final concentration of 1,330 μg / ml,
After keeping at 2 ° C. for 2 hours, the resulting protoplast cells were collected by centrifugation at 4,750 × g for 20 minutes.

The protoplast cells contain 50 mM Tris.
-HCl solution (pH 8.5) 800 ml, 0.5 M E
80 ml of DTA (pH 8) and 80 ml of SLS (sodium sarcosine) (manufactured by Fluka) were added to lyse the cells, and the mixture was allowed to stand at 4 ° C. overnight, and then mixed with an equal volume of phenol / chloroform (1: 1 (v / v)). Mixed with 10,000 ×
The aqueous layer and the organic solvent layer were separated by centrifugation at 20 g for 20 minutes. The operation of mixing the collected aqueous layer with an equal amount of a phenol / chloroform (1: 1 (v / v)) mixed solution and separating the mixture was repeated twice to extract a nucleic acid fraction. An operation of mixing an equal amount of chloroform with the obtained aqueous layer and removing impurities is performed in two steps.
Repeated times. To the obtained aqueous layer, twice the amount of 100% cold ethanol was added to precipitate chromosomal DNA.

Precipitated DNA recovered by winding with a glass rod
Was washed with 100% ethanol, washed and lightly dried. Next, it is dissolved in 100 ml of water, and RNase A is dissolved.
(Boehringer Mannheim) at a final concentration of 100 μg
/ Ml and reacted at 37 ° C for 1 hour to remove mixed RNA. Phenol treatment and chloroform treatment are performed for protein removal, and 400 ml of water is added to the obtained aqueous layer.
Cold ethanol was added, and the reprecipitated DNA was wound up and collected. After dissolving the obtained DNA in 500 ml of water,
50 ml of 3M sodium acetate (pH 5.2) and twice the amount of 100% cold ethanol were added to wind up the precipitated DNA. This operation was repeated twice to remove impurities. The obtained DNA was added with 100% ethanol, washed, and lyophilized.

Embodiment 3 FIG. (Measurement of Component Content of Extracted Substance) The nucleic acid content of the DNA fraction obtained in Example 2 was measured. The nucleic acid was measured using a spectrophotometer (U-2000, manufactured by Hitachi, Ltd.) at a wavelength of 260 nm and 1 OD ₂₆₀ = 20 μg / ml.
It was calculated in terms of DNA. The nucleic acid content of the fraction is 1 m
0.8 mg / l.

1% of the DNA fraction obtained in Example 2 and the DNA fraction of Example 2 which had been treated with enzymes (digested with RNase A and DNase I (Takara Shuzo))
Agarose gel electrophoresis was performed. The result is shown in FIG. The DNA fraction treated with DNase I contains D
No electrophoretic band appeared at the same position as the NA fraction.

20 mg of the DNA fraction of Example 2 was placed in an ampule, 2 ml of 1N-H ₂ SO ₄ was added, the tube was sealed under reduced pressure, and hydrolysis was carried out at 100 ° C. for 18 hours. Thereafter, the container was opened, neutralized with BaCO ₃ , and BaSO ₄ was removed by centrifugation.
10 μl of the treated product was spotted on a thin layer (Whatman silicagel K5), and ethyl acetate: isopropyl alcohol: water =
Thin layer chromatography was performed with a developing solvent of 65:22:11 (v / v / v). Then, after spraying 50% sulfuric acid, 1
After heating at 00 ° C. for 10 minutes, a moving fraction was detected. The results are shown in FIG.

Embodiment 4 FIG. (Hypertensive Effect of Oral Administration of DNA Fraction from Live Enterococcus) Male SHR (15) purchased from Charles River Japan
Weeks) were divided into eight animals per group so that the average body weight of each group was constant. The DNA fraction obtained in Example 2 was dissolved in physiological saline, and 65 mg / kg body weight was orally administered using a gastric tube. The control group received physiological saline. 37
Tail artery systolic blood pressure (mm
Hg) was measured with a non-invasive blood pressure measurement device (MK-1000: Muromachi Kikai Co., Ltd.) immediately before administration, 4, 9, and 24 hours after administration. The results are shown in Table 1 (measured values represent mean ± SD).

[0023]

[Table 1] 時間 Time after administration (h) ─── ────────────────────── 0 492 ──────────────────────── ──────────── Control 237 ± 12 232 ± 7 233 ± 11 232 ± 9 DNA from E. faecalis 240 ± 10 220 ± 9 ** 221 ± 12 * 233 ± 9 ───── ─────────────────────────────── (* p <0.05, ** p <0.01)

Embodiment 5 FIG. (Hypertensive effect of oral administration of calf thymus-derived DNA fraction) Male SHR (15) purchased from Charles River Japan
Weeks) were divided into eight animals per group so that the average body weight of each group was constant. Calf thymus-derived DNA (manufactured by Sigma) was dissolved in physiological saline and orally administered in the same manner as in Example 4. Tail vein systolic blood pressure (mmHg) was measured as in Example 4. The group to which calf thymus-derived DNA was administered was 226.4 4 hours after administration, while the control group was around 240 mmHg.
± 9.1 mmHg, 91.6 hours later, 211.6 ± 15.8 mm
Hg and the value before administration significantly decreased.

Embodiment 6 FIG. (Formulation example) (1) 50 mg of the DNA fraction obtained in Example 2 is mixed with 50 mg of purified starch powder and 200 mg of lactose to prepare tablets or granules.

(2) 100 mg of the DNA fraction obtained in Example 2
Is mixed with 100 mg of soy protein and 200 mg of lactose into tablets or granules.

[0027]

The antihypertensive agent of the present invention has extremely low side effects and toxicity, has a remarkable effect on essential hypertension even when administered orally, has an immediate effect, and has a long-lasting effect. By having these characteristics, it is not limited to use as pharmaceuticals,
It can be added to everyday foods to prevent hypertension. In addition, DNA can be of any origin,
Those that have been discarded, such as animals and fish and shellfish organs (testis and the like) having a high A content, can be effectively used.

[Brief description of the drawings]

FIG. 1 is a diagram showing an agarose gel electrophoresis pattern of a DNA fraction obtained in Example 2 and an enzyme-treated product thereof.

FIG. 2 is a view showing a thin-layer chromatogram of a hydrolyzate of a DNA fraction obtained in Example 2.

──────────────────────────────────────────────────続 き Continued on the front page (58) Field surveyed (Int. Cl. ⁶ , DB name) A61K 31/70 A61K 35/00-35/84 C07H 21/04 WPI (DIALOG)

Claims (3)

(57) [Claims] 1. The method according to claim 1, wherein the active ingredient is deoxyribonucleic acid (DN).
An antihypertensive comprising A). 2. The antihypertensive agent according to claim 1, wherein the deoxyribonucleic acid is obtained from Enterococcus faecalis cells. 3. The antihypertensive agent according to claim 1, wherein the deoxyribonucleic acid is obtained from an organ of an animal such as a thymus.