JPH06703B2 - Antihypertensive - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a medicine, and more particularly to an antihypertensive agent.

[Conventional technology]

Currently, among the diseases with a high mortality rate in Japan, there are extremely many hypertensions or diseases whose underlying diseases are hypertension. Of the hypertension, essential hypertension, whose cause is unknown, accounts for about 90% of the total, and the remaining about 10% is secondary hypertension that accompanies renal, adrenal, and nervous system diseases. It is said that.

The prevention and treatment of hypertension is an important research topic in both basic medicine and clinical medicine because hypertension causes various cardiovascular diseases, and ongoing research is being carried out in various fields. . And many antihypertensive agents have been developed. Antihypertensive drugs currently on the market are roughly classified as follows according to their mechanism of action.

Inhibition of sodium reabsorption in renal tubules Thiazide and other antihypertensive diuretics.

Suppression of storage of noradrenaline Low rolfia alkaloids, etc.

Peripheral vasodilator hydralazine etc.

Adrenergic receptor stimulation Methyldopa, etc.Sympathetic nerve peripheral blockers and sympathetic nerve center depressants, guanethidine, etc., clonidine, etc. β-receptor blockers, propranol, etc. [Problems to be solved by the invention] However, these antihypertensive agents While excellent in antihypertensive action, it also has many side effects. Since antihypertensive drugs are generally used for a relatively long period of time, they are required to be highly safe.

[Means for solving problems]

In view of these present conditions, the present inventors have conducted intensive studies to develop an effective and highly safe drug, which is a plant than conventionally,
The inventors have found that mannan, which is known to be contained in yeast etc., has an excellent antihypertensive effect, and completed the present invention.

That is, the present invention provides an antihypertensive agent containing mannan as an active ingredient.

The mannan used in the present invention refers to a polysaccharide mainly composed of mannose, and for example, the following can be mentioned if classified according to its origin, constitution and the like.

(1) Mannan derived from microorganisms Saccharomyces yeast, red yeast (Rhodotorula glutini
s) yeast, Penicillium (Penicillium)
mold such as charlesii), Bacillus polymyxia (Baci
Mannan obtained from bacterial cells such as llus polymyxa) or the culture solution.

(2) Plant-derived Mannan Elephant palm mannan A, Elephant palm mannan B, red algae mannan, green mannan, orchid mannan in orchid tuber, coffee bean mannan, South African plant huacra
pona palm mannan, Tsukuneimo mannan, yam mannan, etc.

(3) Glucomannan A mannan containing glucose in addition to mannose obtained from rhizomes such as konjac potato, lily, daffodil, and hepatica.

(4) Galactomannan Mannan containing galactose in addition to mannose such as locust bean gum, soybean flesh derived from soybean seed coat, Tamson gum, filamentous fungus galactomannoglycan derived from dermatophyte, and guar gum.

(5) Others Mannan containing two or more kinds of sugars in addition to mannose such as xanthan gum.

Among these mannans, (1) microbial mannan and
(2) Plant-derived mannan, particularly yeast-derived mannan, that is, yeast mannan is preferable.

It has been reported that yeast mannan has many physiological activities. That is, an immunostimulatory action (Japanese Patent Laid-Open No. 51-29215
No.), antitumor effect (JP-A-54-97692, JP-A-58-121216)
No.), anti-infective action (JP-A-58-109423), interferon-inducing activity (E.Matisova 'et al, Acta.Virol., 1
4, 1 (1970)), macrophage migration inhibitory activity (M.Su
zuki & Y. Hayashi, Japan J. Microbiol., 19 , 355 (1975))
Have been reported. However, the effect of yeast mannan and mannan on blood pressure is not known at all.

The mannan used in the present invention can be collected from various microorganisms, plants and the like by a method known per se, but may be a commercially available product (for example, available from Sigma Co., Tokyo Kasei). For example, yeast mannan exists as a cell wall component of yeast, and thus can be extracted from cultured cells.

As yeast, various strains belonging to the genus Saccharomyces,
For example, Saccharomyces ce
revisiae), Saccharomyces dirensis (Saccharo
myces dairensis), Saccharomyces exus (Sac
charomyces exiguus), Saccharomyces kluyveri,
Sa-ccharomyces servazz
ii), Saccharomyces tell
uris), Saccharomyces Unisporus (Saccharomyce
s unis porous). These yeasts are cultivated under culturing conditions adapted to their respective mycological properties, and the microbial cells are collected to obtain cultured microbial cells.

In order to extract yeast mannan from the cultured cells, for example, the cells are subjected to an autoclave heat treatment to obtain a supernatant (autoclave extract), and since mannan is a polysaccharide, the mannan-same complex is obtained by using a filing reagent. It can be carried out by a method of extracting by forming or a deproteinizing method using hydrochloric acid. Further, commercially available yeast extract (for example, sold by Difco Co., Takeda Pharmaceutical Co., Ltd., Daigo Nutrition Co., Ltd.) contains yeast mannan, so it can be used instead of the autoclave extract. .

The autoclave extract was prepared according to the method of Haworth et al. (J. Chem. So.
c., 1937,784), the method of Peat et al. (J. Chem. Soc., 1961,386.
Manufactured according to 1). That is, the cultured cells are suspended in a citrate buffer solution (0.02M, pH 7.0) and heated in an autoclave at 121 ° C for 90 minutes. After cooling, centrifugation is performed and the supernatant is collected. It can be obtained by adding citrate buffer solution to the precipitate portion again, performing the same operation, and combining the obtained supernatant portions.

The extraction / purification method using the Fehling's reagent is carried out as follows. That is, the same amount of the filing reagent is added to the autoclave extract or the yeast extract while stirring. Precipitation of the mannan-copper complex occurs here. The precipitated portion is dissolved in dilute hydrochloric acid, and this is slowly added dropwise to a methanol-acetic acid mixed liquid (8: 1 v / v). Leave for a few hours to collect the mannan precipitate and repeat this operation until the copper color disappears. The precipitate is collected with a glass filter and washed with methanol, ether or the like to obtain crude mannan. Purified mannan can be obtained by dissolving crude mannan in water to remove glucan, adding a filing reagent, and repeating the above operation. Further, after the precipitation of the mannan-copper complex is obtained, it is possible to decopperize it using a strong acid type ion exchange resin such as Amberlite IR120, IR121, IR118 and the like.

The extraction / purification method by deproteinization using hydrochloric acid is carried out as follows. That is, 3N hydrochloric acid was added to the autoclave extract or yeast extract to adjust the pH to 3.5, the precipitates formed were removed, and 3 times the volume of cold methanol was added to the supernatant. The precipitate is collected, suspended in water, and adjusted to pH with 1N caustic soda.
Adjust to 7.0 and remove insolubles. Repeating this operation, it was confirmed that no precipitation occurred at pH 2.5, and the final concentration was 0.5N.
Potassium hydroxide is added so that it becomes, and it is left at 37 ° C. for 1 to 2 hours. The pH is adjusted to 3.5, cold methanol is added 3 times, and the precipitate is suspended in water to adjust the pH to 7.0. This solution is dialyzed against ion-exchanged water overnight. If the removal of proteins and nucleic acids is incomplete, pronase (Nagase & Co., Ltd.), ribonuclease, deoxyribonuclease (Sigma) are added, and after decomposition, the enzyme proteins are removed by the Sebag extraction method.
Dialyze against ion-exchanged water.

The mannan extracted by the above method is further subjected to gel permeation method (Biogel P-300, Biogel A-0.5 m, Sephadex G-200, etc.) and anion exchange chromatography (DEAE-Sephadex A-50, DEAE-cellulose). , DEAE-biogel, etc.) and the like. This purification is carried out, for example, by the method of Okubo et al. (J.
Bacteriol. 136, 63 (1978)).

Of the above extraction methods, the use of copper is problematic in waste liquid treatment and the like, so the method of using hydrochloric acid is preferred.

Analysis value of yeast mannan (hereinafter abbreviated as yeast mannan A) obtained by a method using hydrochloric acid from yeast extract derived from Saccharomyces cerevisiae (Difco) and commercially available yeast mannan (abbreviated as yeast mannan B hereinafter) ) Is shown in Table 1.

In Table 1, sugar was quantified by the phenol sulfate method, protein was quantified by the Lowry method, and phosphorus was quantified by the method of AMES et al.

The antihypertensive action and toxicity of mannan thus obtained will be described below.

I. Antihypertensive Action by Intravenous Administration 1) Method Spontaneously hypertensive rats (SHR; male, 20 weeks old) were used. The SHR was fixed to a table under Nembutal (50 mg / kg or less, intraperitoneal administration) anesthesia. An incision was made in the lateral side of the lower abdomen, a cannula was inserted into the external iliac artery and external iliac vein, and after the incision was closed, the SHR was moved to and fixed on a Ballman type fixing table. The cannula on the arterial side was connected to a blood pressure transducer (manufactured by Nihon Kohden Co., Ltd.), and a physiological saline solution of mannan (10 mg / k as mannan) from the cannula on the vein side.
g) was injected and blood pressure was measured over time.

2) Results For both yeast mannan A and B, hypotension was observed about 3 minutes after intravenous injection. The systolic blood pressure before the vein was 236 mmHg and the diastolic blood pressure was 160 mmHg, but the maximum blood pressure reduction rate was observed 10 to 25 minutes after the intravenous injection of mannan, and the systolic blood pressure at that time was 110 mm.
Hg, diastolic blood pressure was 80 mmHg (hypotension rate: diastolic blood pressure 5
3.4%, minimum blood pressure 50%).

The blood pressure lowering effect of this mannan lasted 1 to 3 hours after intravenous injection.

As a positive control, reserpine (0.13mg
/ kg intramuscular injection), and hydralazine hydrochloride (1 mg / kg IV) were used, hypotension was observed immediately after administration of each drug. In the case of reserpine, blood pressure reduction rate: systolic blood pressure 46.7%, diastolic blood pressure 4
At 6.7%, the effect continued for more than 25 hours after administration.

On the other hand, in case of hydralazine hydrochloride, blood pressure reduction rate;
%, The minimum blood pressure was 53.1%, and its effect continued for more than 25 hours after administration.

II Antihypertensive effect by intraperitoneal administration 1) Method Spontaneously hypertensive rat (SHR; male, 12-14 weeks old)
Was used as one group consisting of 7 animals. As a blood pressure measurement method, a tail artery blood pressure measurement device (Natsume Seisakusho, KN-209) was used, and the SHR was thermostated at 40 ° C. for 13 minutes before blood pressure measurement. Yeast mannans A and B were dissolved in physiological saline and intraperitoneally administered to SHR at 50 and 100 mg / kg. 2, 4, after administration
Blood pressure was measured after 6 and 24 hours. Saline was administered as a control, and α-methyldopa (50, 100 mg / kg) was used as a positive control.

2) Results Changes in systolic blood pressure with time are shown in FIGS. 14% after 2 hours and 20 after 4 hours in the yeast mannan A 50 mg / kg administration group
%, 12% after 6 hours; 23, 20, and 11% in the 100 mg / kg administration group, respectively, compared to before administration. In the yeast mannan B 100 mg / kg administration group, 35.5 and 34.
It had dropped by 0, 19.8%. These antihypertensive effects were similar to those of α-methyldopa.

III. Anti-hypertensive effect by oral administration 1) Method Yeast mannan A and B were dissolved in ion-exchanged water so as to be 200 mg / kg and orally administered using a sonde. Blood pressure was measured.

2) Effect The maximum blood pressure was 2 in the yeast mannan A 200 mg / kg administration group.
4.5% after hours. The value was 7.8% after 4 hours and 6.6% after 6 hours compared with the value before administration. Similarly, in the yeast mannan B 200 mg / kg administration group, it decreased by 7.0%, 8.4% and 6.8%, respectively.

IV, Toxicity Using a ddY mouse (male, 4 weeks old), an acute toxicity test of yeast mannan A and B was performed. Yeast mannan A or B
Was administered intravenously or orally, and the acute toxicity was observed 24 hours later. As a result, the LD ₅₀ values of yeast mannan A and B were both 500 mg / kg or more by intravenous injection and 6 g / kg or more by oral administration.

The antihypertensive agent of the present invention can be a preparation for intravenous administration; a preparation for oral administration such as tablets, capsules, granules and fine granules. In formulating these, in addition to excipients, lubricants, binders and the like usually used as medicines, additives for improving absorption can be added. Although the dose varies depending on the administration method, symptoms, body weight, etc., it is usually preferably 10 to 500 mg / day for an adult, and it is preferable to administer the dose once or several times.

〔The invention's effect〕

The antihypertensive agent of the present invention has an excellent antihypertensive effect, has extremely high safety, and can be continuously used for a long period of time. Therefore, it is useful as a first-line drug for hypertension.

〔Example〕

 Next, the present invention will be described with reference to examples.

Example 1 100 g of yeast extract (Daifuco) was dissolved in 700 ml of ion-exchanged water, and the pH was adjusted to 3.5 using 3N hydrochloric acid.
Insoluble matter generated at this time was centrifuged (3000r.pm, 10
Min.), 3 volumes of cold methanol was added to the resulting supernatant, and the mixture was allowed to stand for about 15 minutes. The resulting precipitate was collected by centrifugation (3000 r.Pm, 10 minutes) and suspended in about 50 ml of ion-exchanged water. The liquid was adjusted to pH 7.0 with 1N-caustic soda, and insoluble matters were removed by centrifugation (18,000 rpm, 10 minutes). The supernatant was adjusted to pH 3.5 with 3N hydrochloric acid and the same operation was repeated 5 times.

Ribonucleolytic enzyme (RNase T ₁ : about 250 units) was added to 30 ml of the finally obtained aqueous solution, and the mixture was reacted at 25 ° C. for 24 hours.

30 ml of a mixture of chloroform / methanol (25/1)
Was added, and the mixture was shaken well and the methanol / water layer was separated. 0.85 g of potassium hydroxide was added to this methanol-water layer, and the mixture was dissolved and shaken at 37 ° C. for 1 hour. The pH was adjusted to 3.5 with 3N hydrochloric acid, 3-fold amount of cold methanol was added, and the precipitate was collected by centrifugation (3,000 rpm, 10 minutes). Suspend it in water,
After adjusting the pH to 7.0 with 1N-caustic soda, it was dialyzed against ion-exchanged water overnight. The dialyzed solution was freeze-dried, dissolved in a small amount of ion-exchanged water, and then applied to Biogel A 0.5 m gel per column. A 0.1N potassium chloride solution was used as an eluent, and the elution of mannan was quantified by the phenol-sulfuric acid method.
Other absorptions at 260 and 280 nm were also measured. Fractions that developed color with phenol-sulfuric acid were collected, dialyzed against ion-exchanged water for 24 hours, and then freeze-dried. By the above series of operations, about 2 g of yeast mannan A was obtained.

Example 2 A tablet having the following formulation was produced.

Yeast Mannan A or B 100 (mg) Dextran 15 Magnesium stearate 2 Lactose 30 Example 3 3 g of yeast mannan A or B was dissolved in 100 ml of physiological saline, and after sterilization, 1 ml was dispensed into ampoules to prepare an injection. .

Example 4 The following components were uniformly mixed and, after sterilization, dispensed in 1 ml aliquots to prepare injections.

Yeast Mannan A or B 3g Distilled water for injection 20ml Polysorbate 0.5g Peanut oil 80ml

[Brief description of drawings]

1 shows physiological saline, FIG. 2 shows α-methyldopa, FIG. 3 shows yeast mannan A, and FIG. 4 shows yeast mannan B.
It is a figure which shows the time-dependent change of the blood pressure at the time of intraperitoneal administration to HR. 1-4, * and ** show the result of a significant difference test. *: 0.01 <P ≦ 0.05 **: 0.001 <P ≦ 0.01

 ─────────────────────────────────────────────────── ─── Continued Front Page (72) Inventor Susumu Ito 3441-64 Higashiminecho, Utsunomiya City, Tochigi Prefecture

Claims (1)

[Claims] 1. An antihypertensive agent containing mannan as an active ingredient.