JPH03255029A - Inhibitor of rise and drop in blood pressure and its production - Google Patents

Abstract

PURPOSE:To obtain an inhibitor for rise and drop of blood pressure, showing effects with a small amount of administration, comprising a water-soluble hemicellulose having a molar ratio of xylose and arabinose in a specific range. CONSTITUTION:An inhibitor for rise and drop of blood pressure, comprising a water-soluble hemicellulose having a saccharide composition with a molar ratio of xylose and arabinose of 1:(0.95-1.2). The preparation is obtained by (i) partially hydrolyzing a water-soluble hemicellulose (preferably with an enzyme), treating the hydrolyzate with an ultrafilter having 8,000-50,000 fractionated molecular weight, separating and recovering a fraction having the above- mentioned saccharide composition, (ii) by dissolving a hemicellulose in a buffer solution having pH 7.5-8.5, passing through an anion exchange resin, absorbing, eluting using an aqueous solution of sodium chloride and recovering a fraction to elute at 0.1-0.3mol concentration and (iii) by adding 80% ethanol solution to a water-soluble hemicellulose and recovering a fraction having the above- mentioned composition from supernatant liquid from which the formed precipitate is separated and removed.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to an agent for lowering and suppressing blood pressure increase, and a method for producing the same.

[Prior Art] In recent years, the effectiveness of dietary fiber has been re-recognized in terms of preventing adult diseases, lowering and suppressing cholesterol rise, regulating the intestines, and preventing colon cancer. As a result, various attempts have been made to actively intake dietary fiber. As an example of such an attempt, it has been proposed to extract hemicellulose from rice bran, wheat bran, etc. and use it as a substance that suppresses increases in serum cholesterol, neutral fat, and liver cholesterol (especially Publication No. 62-6691, Japanese Unexamined Patent Publication No. 62-6691
3-216822, etc.).

[Contents of the Invention] The present inventors have also continued research on improving the physical properties and effective utilization of hemicellulose. Hemicellulose (non-cellulosic polysaccharide) is recovered by removing starch, proteins, lipids, oligosaccharides, inorganic substances, etc. from hemicellulose-containing raw materials such as wheat bran, and extracting the remainder with alkali. The hemicellulose obtained by this alkaline extraction consists of water-soluble hemicellulose and water-insoluble or poorly water-soluble hemicellulose, from which the water-soluble hemicellulose is separated and recovered. Among water-soluble hemicelluloses, water-soluble hemicelluloses derived from wheat bran are generally listed in Table 1 below.
It has the component composition and sugar composition shown in (all % in this specification, including Table 1, means weight %).

[Table 1] Component content of water-soluble hemicellulose derived from wheat bran
MinutesProtein Total SugarOther Approximately 0.1-0.5% Approximately 4.8-10,0% Approximately 86,0-93.6% Approximately 1.4-3.5% TotalSugar Breakdown of the Above Total Sugar ContentXylose Arabinose glucose glucuronic acid Other sugars 100.0% Approximately 45-50% Approximately 35-40% Approximately 2-10% Approximately 3.5-4.5% Approximately O-5% Total 100% It is clear from Table 1 above As shown, water-soluble hemicellulose derived from wheat bran usually has a sugar composition with a xylose:arabinose molar ratio of 1: about 0.80 to 0.89, with the content of xylose being higher than that of arabinose. (Note that both xylose and arabinose have a molecular weight of 1
50, and the comparison by weight % directly becomes the molar ratio).

As a result of research conducted by the present inventors, the water-soluble hemicellulose has a xylose:arabinose molar ratio of 1+0.
．． Classification with sugar composition of 95-1.2 and xylose:
It was confirmed that the molar ratio of arabinose was 1: a mixture of two sugars having a sugar composition of 0.40 to 0.60. The present inventors continued their research to separate and recover the above two categories, and found a smooth separation and recovery method for each category, and found that the molar ratio of xylose:arabinose was 1:0.95 to 1. The category with a sugar composition of .2 exhibits a blood pressure lowering and increase suppressing effect, whereas the category with a sugar composition with a xylose:arabinose molar ratio of 0.40 to 0.60 has a low arabinose content. They also discovered that it does not exhibit the effect of lowering or suppressing blood pressure rise, and completed the present invention.

=4 Therefore, the present invention is a blood pressure lowering and increasing suppressant comprising a water-soluble hemicellulose having a sugar composition with a molar ratio of xylose:arabinose of l:o and a sugar composition of 95 to 1.2.

Further, the present invention includes the following methods (1) to (iii) for producing the blood pressure lowering and increasing suppressing agent.

(i) After partially hydrolyzing water-soluble hemicellulose, it is treated with an ultrafiltration membrane to separate and recover a fraction having a xylose:arabinose molar ratio of l:o and a sugar composition of 95 to 1.2. Characteristic method for producing a blood pressure lowering and increasing suppressant: (ii) After dissolving water-soluble hemicellulose in a buffer solution, it is adsorbed by passing it through an anion exchange resin, and then the concentration of the sodium chloride aqueous solution is adjusted from 0 mol to 0.4 mol. A section having a sugar composition with a gylose:arabinose molar ratio of 1:0.95 to 1.2 is eluted with an eluent having a concentration of 0.1 to 0.3 molar. A method for producing a blood pressure lowering and increasing suppressant, and (iii) adding a 60% ethanol solution to water-soluble hemicellulose and separating and removing a precipitate formed therein. Ratio is 1=0.95
A method for producing a blood pressure lowering and increasing suppressant, which comprises recovering a fraction having a sugar composition of ~1.2.

The water-soluble hemicellulose used as a starting material in methods (i) to (in) above is obtained by extracting bran from wheat, barley, oats, rye, oats, and other hemicellulose-containing raw materials with an alkali. It is produced by separating and collecting the water-soluble fraction from the hemicellulose obtained there. Any water-soluble hemicellulose may be used without limitation.

In producing the agent for lowering and suppressing blood pressure increase of the present invention by the method (i) above, first, the water-soluble hemicellulose described above is partially hydrolyzed. Through this partial hydrolysis treatment, a classification (hereinafter referred to as
3 categories) are selectively hydrolyzed to produce xylose:
The segment having a sugar composition with an arabinose molar ratio of 1+0.95 to 1.2 (hereinafter referred to as segment) remains almost unhydrolyzed. Partial hydrolysis of water-soluble hemicellulose is carried out using enzymes, acids, alkalis, etc., and treatment with enzymes is preferable because it does not involve excessive or insufficient hydrolysis, and the desired substance can be efficiently produced under mild conditions. As the enzyme for this purpose, plant fiber tissue disintegrating enzymes such as hemicellulase and cellulase can be used. At this time, the enzyme may be used in a free state or immobilized on a carrier. Further, the partial hydrolysis treatment may be carried out in a continuous method or in a batch method. Depending on the type of water-soluble hemicellulose, whether the enzyme is used in a free or immobilized state, whether it is carried out in a continuous method or a batch method, etc., the type of enzyme, its usage amount, and the time of partial hydrolysis are determined. It is preferable to select the temperature, pressure, pH, etc. for 1 hour.

- For example, when partially hydrolyzing water-soluble hemicellulose derived from wheat bran using hemicellulase (Onozuka R3''' manufactured by Yakult), approximately 1 to 100% of the hemicellulose
Form a 10% aqueous solution (pH about 3.5 to 7.0), add about 2 to 1001000 pp/v of 1' Onozuka RS'' to this aqueous solution, and heat at a temperature of about 40-60°C for about 5 minutes.
It is recommended to hydrolyze for 1 hour. In this hydrolysis reaction, the viscosity of the hemicellulose aqueous solution initially decreases rapidly and then gradually decreases, but it is preferable to stop the hydrolysis reaction when the rapid viscosity decrease ends. Next, the partially hydrolyzed water-soluble hemicellulose aqueous solution is treated with an ultrafiltration membrane to separate and recover the above-mentioned Class A. As the ultrafiltration membrane used here, any of the conventionally known membranes made of polymers such as polysulfone, acrylonitrile copolymer, aromatic polyamide, polyvinylidene fluoride, polyethersulfone resin, and polyimide resin can be used. can. In addition, there are various shapes known, such as tubular membranes, flat membranes, spiral modules, and hollow modules, among which polysulfone tubular membranes with an inner diameter of 10 to 15 mm are used to prevent clogging. This is preferable because a large amount of aqueous solution can be purified in a short period of time. Polyolefin-based ultrafiltration membranes are undesirable because they tend to adsorb target substances. The fractionation performance of the membrane is approximately 8,000 to 50 when expressed in molecular weight cutoff.
000 is preferred. When separating and recovering the target product using this ultrafiltration membrane, an aqueous solution of partially hydrolyzed water-soluble hemicellulose is passed through the inside of the tubular membrane and filtered, and a permeate containing substances other than the target product appears on the outside of the membrane. Process to discharge. At this time, the target product can be efficiently obtained by constantly supplying the same amount of water to the inside of the tubular membrane as the amount of solution passing through the membrane to keep the amount of membrane treatment liquid constant. In that case, the aqueous solution inside the tubular membrane usually contains about 5 to 10
The treatment is carried out by applying a pressure of 1.5 kg-f/cm. It is also preferable to maintain the temperature of the aqueous solution at about 40 to 60° C. during the treatment.

When treated with an ultrafiltration membrane in this manner, Class A is obtained.

In addition, when using the method (■) above, the pH is about 7.5 to
A solution of about 150-250 mg of water-soluble hemicellulose per 20 ml of 8.5 m buffer is adsorbed through an anion exchange resin, and then the buffer is absorbed at 0.5-1 m
．． The solution was passed at a rate of 5 mQ/min for 5 to 6 hours, and then the sodium chloride concentration of the eluent consisting of an aqueous sodium chloride solution was gradually increased from 0 mol to about 0.4 mol, while increasing the sodium chloride concentration from 0.5 to 1.
．． When the ion-exchange resin adsorbate is eluted by passing at a rate of 5 mQ/min, a large fraction is selectively eluted in the eluted fraction obtained from the eluent with a thorium chloride concentration of 0.1 to 0.3 mol. It is collected and desalted. In the case of using such an ion exchange resin, Tris-HCl buffer, phosphate buffer, etc. can be used as the above-mentioned buffer solution, and as an anion exchange resin, DEAE combined with a cellulose derivative can be used. , especially DEAE-3
Epharose (manufactured by Pharmacia, Sweden) is preferred.

Furthermore, in the case of the above method (Ichi), ethanol is added to a solution in which about 10 to 100 g of water-soluble hemicellulose is dissolved per 1Q of water, and when the ethanol solution is adjusted to 60%, 8 sections will precipitate, so 60% The large fraction dissolved in the ethanol solution was collected as a supernatant liquid, and
% ethanol solvent can be removed by any method to recover the target product.

The component composition, sugar composition, and 5% aqueous solution of the water-soluble hemicellulose as the starting material, the enzymatic partial hydrolyzate, and the antihypertensive and antihypertensive agent of the present invention are as shown in Table 2 below. .

11- 2 Ash Minute (%) protein(%) Total sugar amount (%) others(%) total (%) Total sugar measurement breakdown Xylose (%) Arabinose (%) Glucose (%) Glucuronic acid (%) Other sugars (%) total(%) 5% aqueous solution viscosity (cP) [Table-2] 0.1-0.5 4.8~10.0 86.0-93.6 1.4-3.5 ioo,.

45-50 35-40 2-10 3.5-4.5 0-5 100.0 +68 0.1-0.5 4.5-10.0 85.0-95.0 1.2-3. 5 100.0 45-50 35-40 2-10 3.5-4.5 0-5 100.0 4 0.1-0.5 4.5-10.0 85, O-95.0 1. 2-3.5 100.0 40-45 40-45 0-5 4.5-5.5 0-5 1.00.0 0 Here, the total sugar amount in Table 2 above is calculated as follows. I asked. Each component in sugar (xylose, arabinose,
The content of hemicellulose (glucose, etc.) is Ti1O,
The method of BHATTI et al. [Biochim, Biop
hys, Acta, 222 (1970) 339
-347], followed by methylation and analysis using gas chromatography. All values in Table 2 are values calculated as dry matter.

[Method for measuring total sugar content] The aqueous solution containing sugars is diluted 100 times with distilled water.

Next, 0.5 m4 of a 5% phenol aqueous solution was added to 0.5 m4 of this diluted aqueous solution (2), and after stirring, 3 mO concentrated sulfuric acid was added.
, and further stirred. After leaving it as it is for 20 minutes and cooling it in the air, absorbance at a wavelength of 490 nm is measured. The total sugar content was determined by comparing the measured values with a drift curve using xylose as a substrate.

The antihypertensive and antihypertensive agent of the present invention is a water-soluble white powder, and by administering it to humans and various animals (for example, pets such as dogs and cats) in small amounts, it can be used to treat hypertensive humans. It is possible to lower the blood pressure of animals and animals, and furthermore, it is possible to suppress the increase in blood pressure.

Furthermore, the agent for lowering and suppressing blood pressure increase of the present invention may contain potassium, which is known to be involved in lowering blood pressure and suppressing blood pressure increase, and the combined use of potassium further enhances the effect of lowering blood pressure and suppressing blood pressure increase. be done. Potassium in this case is preferably used in the form of an inorganic potassium salt such as potassium chloride, potassium carbonate, potassium phosphate, or an organic potassium salt.

The preferred dosage of the antihypertensive and antihypertensive agent of the present invention is as follows:
Although it naturally varies depending on various conditions such as the age, weight, sex, symptoms, and type of animal of the human or animal to be administered, for example,
For humans, it is approximately 0.01 to 0.0.0% per kg of body weight per day.
It can be administered at a rate of 5 g, or in animals at a rate of about 0.1 to 10 g per kg body weight per day.

The agent for lowering and suppressing blood pressure increase of the present invention is administered orally. Furthermore, the antihypertensive agent of the present invention, etc.
They may be administered alone, together with solid or liquid carriers commonly used in the pharmaceutical industry, or mixed with or in combination with other agents. The dosage form can be any form such as tablets, pills, granules, capsules, powders, and aqueous solutions.

Furthermore, the agent for lowering and suppressing blood pressure increase of the present invention can be added to or administered together with foods and feeds.

The present invention will be specifically explained below by giving examples, but the present invention is not limited thereto.

Reference Example [Preparation of water-soluble hemicellulose derived from wheat bran] Disperse 2 kg of selected wheat bran in 20Q of warm water at 50°C and stir for 5 minutes. After that, the solid content was collected by filtration using a centrifugal filter (Tanabe Iron Works), and 3 kg of the obtained solid content was heated at 70°C and
．． Add to 2N aqueous sodium hydroxide solution 2012 and stir for 90 minutes. After cooling, 0.8N hydrochloric acid aqueous solution 5a is gradually added under stirring for neutralization. Centrifuge the neutralized solution at 5,0 OOG for 10 minutes. The supernatant liquid is separated, diluted with water to a total sugar content of 5 mg/mQ, and kept at a temperature of 50°C.

The entire solution was filtered through a tubular ultrafiltration membrane (manufactured by Nitto Denko: NTU352).
0PI8 type, membrane area 0-76 m”, inner diameter 1.1.5 m
m), pressure 8 kg/cm", flow rate 13 Q
/min for 3 hours. At this time,
The same amount of water as the membrane permeation solution is always replenished into the tube to keep the amount of membrane treatment liquid constant. Stop the supply of drained water for 3 hours, start concentration under the same conditions as above, concentrate without considering the decrease in flux, and continue for about 1.5 hours until the sugar concentration of the aqueous solution reaches about 1゜n+g/mQ. conduct. The treated solution is eluted into a 500 cc cation exchange resin IR-120E manufactured by Organo Co., Ltd. at a flow rate of 10 times the exchange capacity of the ion exchange resin per hour, and then passed through an anion exchange resin IRA-93 manufactured by the same company at the same flow rate.

After the ion exchange treatment, the resulting aqueous solution was vacuum freeze-dried at a temperature of 30° C., vacuum level Q, and 1 Torr or less) to obtain 150 g of white hemicellulose powder (a) (recovery rate divided into selected wheat bran = 7. 5%). The obtained hemicellulose powder 6 powder (a) was completely dissolved in water at 25°C.

Example 1 A 1% aqueous solution was prepared by dissolving 150 g of water-soluble hemicellulose powder derived from wheat bran (A) prepared in the two reference examples in 15 ffl of an acetate buffer aqueous solution (acetate content 50 mmol, pH 5.5). did.

After preheating this solution for 40° C. for 10 minutes, 75 μg of hemicellulase (“Onozuka R3” manufactured by Yakult) was added, and partial hydrolysis was carried out for 20 minutes while maintaining the same temperature. Thereafter, the solution was heated at 9,000° C. for 10 minutes to inactivate the enzyme. A portion (1.0 ml) of this liquid was collected and freeze-vacuum dried at 25° C. at 110 mTorr to obtain 0.47 gt of hemicellulose powder (b).

In addition, while maintaining the temperature of the remainder of the above partial hydrolysis solution at 50°C, a tubular ultrafiltration membrane (NTU3520-PI3 type: manufactured by Nitto Denko Corporation: membrane area 0.76 m', inner diameter 11.5 mm) was added.
through the pipe at a pressure of 8 kg-f/cm2 and a flow rate of 13 (1
/ min conditions. At this time, the same amount of water as the membrane permeation solution was constantly replenished into the tube to keep the amount of membrane treatment liquid constant. After 3 hours, the water supply was stopped and concentration was started under the same conditions as above, and concentration was carried out until the sugar concentration of the aqueous solution reached about 25 mg/m4 without considering the decrease in flux. The obtained concentrate was freeze-dried under vacuum at 25° and 0.100 mTorr to obtain 102 g of white hemicellulose powder (c).

Example 2 Ethanol was added to a solution in which 150 g of the water-soluble hemicellulose derived from wheat bran (A) prepared in the above reference example was dissolved in 2Q of water to adjust the ethanol concentration to 60%, and the solution was stirred for about 20 minutes and then allowed to stand still. .

The precipitate was washed with 60% ethanol and freeze-dried in vacuum to obtain 60 g of a white product (d).

In addition, 90 g of a white product (e) was obtained by evaporating the ethanol component of the filtrate (supernatant liquid) using an evaporator and then freeze-vacuum drying it at 25°C and 100 mTorr.
I got it.

Example 3 200 mg of water-soluble hemicellulose derived from wheat bran (A) prepared in the above reference example was dissolved in 20 mmol of Tris-HCl buffer (pH 8,5) 20 mΩ, and this solution was mixed with anion exchange resin DEAE-5epharose CL-6B. (
The mixture was passed through a column packed with 150 mQ (manufactured by Pharmacia) to adsorb it, and then the same buffer was passed through it at a flow rate of 0.7 mQ 1 minute for 5 hours to wash away non-adsorbed substances. Next, an eluent with a sodium chloride concentration gradually increasing from 0 to 0.4 mol was passed through the column at a flow rate of 0.7 mQ1 min.
An elution fraction obtained from 0.3 mol of eluent was obtained. When the liquid containing the non-adsorbed matter and the liquid containing the adsorption/elution section were each dried, white hemicellulose powder (non-adsorbed material) and white hemicellulose powder (adsorbed material) were obtained at a weight ratio of 40+60. (G) were obtained.

The water-soluble hemicellulose obtained in the above reference example (a), the hydrolyzed hemicellulose of Example 1 (b) and the ultrafiltration membrane-treated hemicellulose (c), the precipitated hemicellulose obtained in Example 2 (d) and the supernatant hemicellulose (ho). ), and the content of each component in the hemicellulose (non-adsorbed material) (g) and hemicellulose (adsorbed material) (g) of Example 3 are as shown in the following table -319-20- there were.

Table 1-3] lf lo ha ni ho he
Protein (%) 5.3 4.7 5.0 2.5
5.4 0.8 4.8 Sugars (%') 91
．． 0 B9.7 91.8 93.6 90.4 9
6.4 92.4 Example 4 Test foods 1 to 2 having the compositions shown in Table 4 below were prepared.

[Table J Casein L-medionine Mineral Mix Vitamin Mix Sugar Lad Salt Reference Example Water Soluble Hemicellulose (A) 3.8 Ultrafiltration Membrane Treated Hemicellulose (C) of Example 1 3.7 , In the above test foods ■ to ■, the water-soluble hemicellulose (A) of the reference example and the ultrafiltration membrane-treated hemicellulose of Example 1 were used so that the total amount of xylose and arabinose in the test food was 3%. (c) and the precipitated hemicellulose (d) of Example 2 are each blended.

Next, four groups of spontaneously hypertensive rats (male, 9 weeks old, average weight 280 g/animal), 6 rats in each group, were prepared.

Two doses of each of the test foods 1 to ■ above were given to the rats in each group.
They were given free access for a week.

The blood pressure of the rats in each group was measured initially, one week later, and two weeks later, and the results were as shown in Table 5 below.

[Table-5] Blood pressure (mm) Ig) Initially 1 week after 2 weeks 141±7.5 140±8.5 140±9.6 1
41±6.6153±4.0 147±4.6 1.4
6±19.2 152±4.3170±7.6 160
±5.8 * 158 ± 5.3 * 167 ± 6.0 The increase in blood pressure was hardly suppressed in the rats in group ■ that were fed the test food containing the water-soluble hemicellulose (A) of the reference example and the ultrafiltration membrane-treated hemicellulose (C) of Example 1. It can be seen that the increase in blood pressure was suppressed in the rats in the ■ and ■ groups containing the drug, and the suppression of the increase in blood pressure was particularly large in the ■ group.

From this, it can be seen that hemicellulose consisting of category A is effective in suppressing an increase in blood pressure. Also, ■
The water-soluble hemicellulose (A) used in Section 8 is
It is thought that the phytochemical contains both of the two categories, and that the subdivision contained therein acts as an antihypertensive agent.

[Effects of the Invention] The agent for lowering and suppressing blood pressure increase of the present invention exhibits a blood pressure lowering effect and a blood pressure increase suppressing effect when administered in a small amount.

Moreover, when using the production method of the present invention, highly pure blood pressure lowering and increasing suppressant 23 4- can be smoothly obtained with simple operations.

Claims (1)

[Claims] 1) The molar ratio of xylose:arabinose is 1:0.95
A blood pressure lowering and increasing suppressant comprising water-soluble hemicellulose having a sugar composition of ~1.2. 2) After partially hydrolyzing water-soluble hemicellulose, it is treated with an ultrafiltration membrane to separate and collect a segment having a sugar composition with a xylose:arabinose molar ratio of 1:0.95 to 1.2. The method for producing the agent for lowering and suppressing blood pressure increase according to claim 1. 3) After dissolving the water-soluble hemicellulose in a buffer solution, it was passed through an anion exchange resin to adsorb it, and then passed through an eluent in which the concentration of sodium chloride was gradually increased from 0 mol to 0.4 mol. The molar ratio of xylose:arabinose is 1:0.95, which is eluted with an eluent of .1 to 0.3 mol.
The method for producing a blood pressure lowering and increasing suppressant according to claim 1, characterized in that a fraction having a sugar composition of 1.2 to 1.2 is recovered. 4) A 60% ethanol solution was added to the water-soluble hemicellulose, and the resulting precipitate was separated and removed. From the supernatant, the molar ratio of xylose:arabinose was 1:0.95 to 1.
2. The method for producing a blood pressure lowering and increasing suppressant according to claim 1, wherein a fraction having a sugar composition of 2 is recovered.