JP2804587B2 - Antihypertensive / elevation inhibitor and production method thereof - Google Patents

Description

Description: FIELD OF THE INVENTION The present invention relates to an antihypertensive and antihypertensive agent, and a method for producing the same.

[Prior art] In recent years, the effectiveness of dietary fiber (dietary fiber) has been re-recognized in terms of prevention of adult diseases, suppression of cholesterol lowering and elevation, intestinal control, prevention of colorectal cancer, and the like. Accompanying this, various attempts have been made to actively ingest dietary fiber. As an example of such an attempt, it has been proposed that hemicellulose is extracted from rice bran or wheat husum and used as an inhibitor of serum cholesterol, neutral fat, and hepatic cholesterol elevation (particularly). JP-B-62-6669, JP-A-63-21
No. 6822).

[Contents of the Invention] The present inventors have also continued research on improvement of physical properties and effective utilization of hemicellulose. Hemicellulose (non-cellulosic polysaccharide) is obtained by first removing starch, proteins, lipids, oligosaccharides, and minerals from hemicellulose-containing raw materials such as bran,
The remainder is recovered by extraction treatment with alkali.
The hemicellulose obtained by this alkali extraction is composed of water-soluble hemicellulose and water-insoluble or hardly water-soluble hemicellulose, from which water-soluble hemicellulose is separated and recovered. Among the water-soluble hemicelluloses, the water-soluble hemicellulose derived from bran is generally listed in the following Table-
It has the component composition and sugar composition shown in Table 1 (all percentages in the present specification including Table 1 mean weight%).

As is evident from Table 1 above, bran-derived water-soluble hemicellulose usually has a sugar composition in which the molar ratio of xylose: arabinose is 1: about 0.80 to 0.89, and xylose has a higher content than arabinose. (Note that both xylose and arabinose have a molecular weight of 150, and the molar percentage is equivalent to the percentage by weight.)

As a result of studies by the present inventors, the water-soluble hemicellulose has a xylose: arabinose molar ratio of 1: 0.
It was confirmed that the mixture consisted of a mixture having a sugar composition of 95 to 1.2 and a mixture having a sugar composition of xylose: arabinose having a molar ratio of 1: 0.40 to 0.60. And
The present inventors continued their research to separate and recover the above two sections, and found a method of smooth separation and recovery of each section, and the above xylose: arabinose molar ratio was 1: 0.
The section having a sugar composition of 95 to 1.2 exhibits a blood pressure lowering and elevation suppressing action, whereas the above xylose: arabinose molar ratio of less arabinose content is 1: 0.40 to 0.60.
The present inventors have also found that a category having a sugar component does not exhibit a blood pressure lowering or raising effect and completed the present invention.

Therefore, the present invention is an antihypertensive and antihypertensive agent comprising water-soluble hemicellulose having a sugar composition having a molar ratio of xylose: arabinose of 1: 0.95 to 1.2.

The present invention also includes the following methods (i) to (iii) for producing the blood pressure lowering and increasing inhibitor.

(I) After partially hydrolyzing the water-soluble hemicellulose,
A method for producing an antihypertensive or antihypertensive agent, comprising treating with an ultrafiltration membrane and separating and recovering a fraction having a saccharide composition of xylose: arabinose having a molar ratio of 1: 0.95 to 1.2, (ii) aqueous solution After dissolving the soluble hemicellulose in the buffer,
Adsorbed through an anion exchange resin, then passed through an eluent in which the concentration of an aqueous sodium chloride solution was gradually increased from 0 mol to 0.4 mol, and eluted with an eluent having a concentration of 0.1 to 0.3 mol xylose: arabinose mole Ratio 1: 0.95-1.
2. A method for producing an antihypertensive or antihypertensive agent, comprising recovering a section having a saccharide composition of (2), and (iii) adding a 60% ethanol solution to water-soluble hemicellulose, separating and removing a precipitate formed therefrom. A method for producing an antihypertensive or antihypertensive agent, comprising recovering a fraction having a saccharide composition of a xylose: arabinose molar ratio of 1: 0.95 to 1.2 from a supernatant.

In the methods (i) to (iii), the water-soluble hemicellulose used as a starting material is wheat, barley, oat,
It is produced by subjecting barley bran such as rye and oats and other hemicellulose-containing raw materials to an extraction treatment with alkali, and separating and recovering a water-soluble fraction from the obtained hemicellulose. Any water-soluble hemicellulose may be used and is not limited thereto.

Then, in producing the blood pressure lowering and increasing inhibitor of the present invention by the method (i), the above-mentioned water-soluble hemicellulose is first partially hydrolyzed. By such a partial hydrolysis treatment, a section having a saccharide composition of a xylose: arabinose molar ratio of 1: 0.4 to 0.6 (hereinafter referred to as B section) is selectively hydrolyzed, and a xylose: arabinose molar ratio of 1: 0.95. A section having a sugar composition of 1.2 (hereinafter, referred to as section A) remains almost without hydrolysis.
The partial hydrolysis is performed on water-soluble hemicellulose with an enzyme, an acid, an alkali, or the like. However, the treatment with the enzyme is preferable since the desired substance can be efficiently produced under mild conditions without excessive or insufficient hydrolysis. As an enzyme for that,
Plant fiber tissue-disintegrating enzymes such as hemicellulose and cellulase can be used. At that time, the enzyme may be used in a free state or immobilized on a carrier. The partial hydrolysis treatment may be performed by a continuous method or a batch method. Depending on the type of water-soluble hemicellulose, whether the enzyme is used in a free state or immobilized, is used in a continuous method, or is performed in a batch method, etc., the type of the enzyme, the amount used, and during partial hydrolysis The temperature, pressure, pH, time, etc. should be selected. For example, when the water-soluble hemicellulose derived from wheat bran is partially hydrolyzed using a hemicellulase ("Onozuka RS" manufactured by Yakult Co., Ltd.), an about 1 to 10% aqueous solution (pH about 3.5 to 7.0) of the hemicellulose is formed. About 2 to 1000 ppm (w / v) of "Onozuka RS" is added to
Hydrolysis at a temperature of 〜60 ° C. for about 5 minutes to 1 hour is preferred. In this hydrolysis reaction, the viscosity of the hemicellulose aqueous solution decreases sharply initially and then gradually decreases,
It is preferable to stop the hydrolysis reaction at the time when the rapid decrease in viscosity is completed. Next, the aqueous solution of partially hydrolyzed water-soluble hemicellulose is treated with an ultrafiltration membrane to separate and collect the above section A. As the ultrafiltration membrane used here, any of conventionally known membranes made of polymers such as polysulfone, acrylonitrile copolymer, aromatic polyamide, polyvinylidene fluoride, polyether sulfone resin, and polyimide resin are used. it can. In addition, tubular membranes, flat membranes, spiral modules, hollow modules, and other shapes are known due to differences in shape. It is preferable because a large amount of aqueous solution can be purified in a long time. Polyolefin-based ultrafiltration membranes are not preferred because they tend to cause the adsorption of the target substance. The fractionation performance of the membrane is preferably about 8,000 to 50,000 in terms of the fractional molecular weight. When separating and recovering the target substance by the ultrafiltration membrane, the aqueous solution of the partially hydrolyzed water-soluble hemicellulose is passed through the inside of the tubular membrane to perform filtration, and the permeate containing substances other than the target substance is put on the outside of the membrane. Perform the process of discharging. At that time, if the same amount of water as the amount of the solution permeating the membrane is always supplied to the inside of the tubular membrane to carry out the treatment while keeping the amount of the membrane treatment liquid constant, the desired product can be obtained efficiently. In that case,
The treatment is performed by applying a pressure of about 5 to 10 kg · f / cm ² to the aqueous solution inside the tubular membrane. In addition, the temperature of the aqueous solution during processing is about 40-60 ° C.
It is better to keep it.

When the ultrafiltration membrane treatment is performed by such a method, the A category is obtained.

In the case of the above method (ii), the pH is about 7.5 to 8.5.
About 150-250 mg of water-soluble hemicellulose per 20 ml of buffer
Is adsorbed through an anion exchange resin, and then the buffer is passed at 0.5 to 1.5 ml / min for 5 to 6 hours. When eluted with ion-exchange resin adsorbed by passing the solution at 0.5 to 1.5 ml / min while gradually increasing from 0.4 mol to about 0.4 mol, the sodium chloride concentration was 0.1 to 0.3.
The A segment is selectively eluted in the elution segment obtained from the molar eluate, and is recovered and desalted. In the case of using such an ion exchange resin, a Tris-HCl buffer, a phosphate buffer, or the like can be used as the buffer, and a resin obtained by binding a cellulose derivative to DEAE can be used as the anion exchange resin. , Especially DEAE-Se
pharose (Pharmacia, Sweden) is preferred.

Further, in the case of the above method (iii), ethanol is added to a solution in which about 10 to 100 g of water-soluble hemicellulose is dissolved per water, and the B solution for adjusting the ethanol solution to 60% precipitates. The Class A dissolved in the ethanol solution is recovered as a supernatant, and the 60% ethanol solvent is removed by an arbitrary method to recover the target substance.

The starting material, water-soluble hemicellulose, the partial hydrolysis by the enzyme and the component composition, sugar composition and 5% aqueous solution of the antihypertensive / hypertensive agent of the present invention are almost as shown in Table 2 below.

Here, the total sugar amount in Table 2 above was determined as follows. The content of each component (xylose, arabinose, glucose, etc.) in the sugar is determined by converting hemicellulose into TARIO,
BHATTI et al. [Biochim. Biophys. Acta, 222 (1970) 33
9 to 347], followed by methylation, which was determined by analysis using gas chromatography. All the values in Table 2 are values converted as dry matter.

[Measurement Method of Total Sugar Content] An aqueous solution containing a saccharide is diluted 100 times with distilled water.
Next, 0.5 ml of a 5% phenol aqueous solution was added to 0.5 ml of the diluted aqueous solution, and the mixture was stirred, and then 3 ml of concentrated sulfuric acid was added, followed by further stirring. Leave it for 20 minutes to cool it down,
Measure the absorbance at 0 nm. The measured value was compared with a standard curve using xylose as a substrate to determine the total sugar amount.

The antihypertensive and antihypertensive agent of the present invention is a white powder of an aqueous solution, which is used for humans and various animals (for example,
Pets such as dogs and cats)
The blood pressure of humans and animals with hypertension can be reduced, and the increase in blood pressure can be suppressed.

The antihypertensive and antihypertensive agent of the present invention may contain potassium which is known to be involved in hypotensive or antihypertensive treatment, and the combined use of potassium further enhances the antihypertensive and antihypertensive effects. Is done. In this case, potassium is preferably used in the form of an inorganic potassium salt such as potassium chloride, potassium carbonate, potassium phosphate or the like or an organic potassium salt.

The preferred dosage of the antihypertensive and antihypertensive agent of the present invention is the age, weight, sex, symptoms, and age of the human or animal to be administered.
Naturally, it varies depending on various conditions such as the type of animal. For example, in the case of humans, at a rate of about 0.01 to 0.5 g per kg of body weight per day, and in the case of animals, about 0.1 to 0.5 g per kg of body weight per day.
It can be administered at a rate of 10 g.

And the antihypertensive and antihypertensive agent of the present invention is administered orally. Further, the antihypertensive agent of the present invention may be administered alone, may be administered together with a solid carrier or liquid carrier usually used in the pharmaceutical industry, or may be mixed with a drug resistant to be used. , Can also be used in combination. The dosage forms are tablets, pills, granules,
It can be used in any form such as capsule, powder, aqueous solution and the like.

Further, the antihypertensive and antihypertensive agent of the present invention can be added to foods and feeds and administered together therewith.

Hereinafter, the present invention will be specifically described with reference to examples, but the present invention is not limited thereto.

Reference example [Preparation of water-soluble hemicellulose derived from wheat bran] 2 kg of carefully selected wheat bran is dispersed in warm water 20 at 50 ° C and stirred for 5 minutes. Thereafter, the solid content was collected by filtration with a centrifugal filter (manufactured by Tanabe Iron Works), and 3 kg of the obtained solid content was filtered at 70 ° C. and 0.2N
Stir in sodium hydroxide aqueous solution 20 for 90 minutes. After the law, 0.8N hydrochloric acid aqueous solution 5 is gradually added with stirring to neutralize. Centrifuge the neutralization solution at 5,000 G for 10 minutes. The supernatant is collected, diluted with water so that the total sugar content is 5 mg / ml, and kept at a liquid temperature of 50 ° C. The whole solution was subjected to a tubular ultrafiltration membrane (Nitto Denko: NTU3520-P18 type, membrane area 0.76 m ² , inner diameter 11.5 m
m), and treated under the conditions of a pressure of 8 kg / cm ² and a flow rate of 13 / min for 3 hours. At this time, the same amount of water as the membrane permeating solution is always replenished into the tube, and the amount of the membrane treatment solution is kept constant. After 3 hours, supply of water is stopped, concentration is started under the same conditions as described above, concentration is performed without considering a decrease in flux, and the concentration is performed for about 1.5 hours until the sugar concentration of the aqueous solution becomes about 10 mg / ml. The treated solution is eluted at a flow rate of 10 times the ion exchange resin exchange capacity per hour into 500 cc of a cation exchange resin IR-120E manufactured by Organo Corporation, and then flown at the same flow rate into an anion exchange resin IRA-93 manufactured by the company. After the ion exchange treatment, the resulting aqueous solution is freeze-dried in a vacuum (temperature
150 g of white hemicellulose powder (a) was obtained at 30 ° C. and a degree of vacuum of 0.1 Torr or less (recovery rate for selected wheat bran:
7.5%). The obtained hemicellulose powder (a) was completely dissolved in water at 25 ° C.

Example 1 A 1% aqueous solution was prepared by dissolving 150 g of the bran-derived water-soluble hemicellulose powder (a) prepared in the above reference example in an acetate buffer aqueous solution (acetate content: mmol, pH 5.5) 15.

After the solution was previously heated at 40 ° C. for 10 minutes, 75 μg of hemicellulase (“Onozuka RS” manufactured by Yakult) was added, and partial hydrolysis was performed at the same temperature for 20 minutes. afterwards,
The solution was heated at 90 ° C. for 10 minutes to inactivate the enzyme. A portion (10 ml) of this solution was collected and freeze-vacuum dried at 25 ° C. and 100 mTorr to obtain 0.47 g of hemicellulose powder (b).

While maintaining the temperature of the remaining part of the partial hydrolyzate at 50 ° C., a pressure of 8 kg was passed through a tube of a tubular ultrafiltration membrane (NTU3520-P18 type: manufactured by Nitto Denko Corporation: membrane area 0.76 m ² , inner diameter 11.5 mm).
The treatment was performed under conditions of f / cm ² and a flow rate of 13 / min. At this time,
The same amount of water as the membrane permeating solution was always supplied into the tube to keep the amount of the membrane treatment solution constant. After 3 hours, the water supply was stopped and the concentration was started under the same conditions as described above, and the concentration was performed until the sugar concentration of the aqueous solution became about 25 mg / ml without considering the decrease in the flux.
The resulting concentrate was freeze-vacuum dried at 25 ° C. and 100 mTorr to obtain 102 g of white hemicellulose powder (c).

Example 2 Ethanol was added to a solution prepared by dissolving 150 g of the bran-derived water-soluble hemicellulose (a) prepared in the above reference example in water 2, and the ethanol concentration was adjusted to 60%.
After stirring for 20 minutes, it was left still.

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

The filtrate (supernatant) was evaporated with an ethanol component using an evaporator, and then freeze-vacuum dried at 25 ° C. and 100 mTorr to obtain 90 g of a white product (e).

Example 3 200 mg of the bran-derived water-soluble hemicellulose (a) prepared in the above Reference Example was added to a 20 mM Tris-HCl buffer (pH
8.5) Dissolve in 20 ml, and add this solution to the anion exchange resin DEAE-S
The adsorbed solution was passed through a column packed with 150 ml of epharose CL-6B (manufactured by Pharmacia) and adsorbed. Then, the same buffer solution was passed at a flow rate of 0.7 ml / min for 5 hours to wash away non-adsorbed substances. Next, an eluent having a gradually increasing sodium chloride concentration from 0 to 0.4 mol was passed through the column at a flow rate of 0.7 ml / min to obtain an elution fraction obtained from an eluent having a concentration of 0.1 to 0.3 mol. When the liquid containing the non-adsorbed substance and the liquid containing the adsorption-elution section were dried, a white hemicellulose powder (non-adsorbed substance) (f) and a white hemicellulose powder (adsorbed) in a weight ratio of 40:60 were obtained. (G) were obtained.

The water-soluble hemicellulose (a) obtained in the above reference example, the hydrolyzed hemicellulose (b) of Example 1 and the hemicellulose treated with an ultrafiltration membrane (c), the precipitated hemicellulose (d) obtained in Example 2, and the supernatant hemicellulose (e) ), And the content of each component in the hemicellulose (non-adsorbed substance) (f) and the hemicellulose (adsorbed substance) (g) of Example 3 were as shown in Table 3 below.

Example 4 Test meals I to IV having the compositions shown in Table 4 below were prepared.

In the test meals II to IV, the total content of xylose and arabinose in the test meal was 3%,
Each of the water-soluble hemicellulose (a) of the reference example, the hemicellulose treated with the ultrafiltration membrane (c) of Example 1, and the precipitated hemicellulose (d) of Example 2 was blended.

Subsequently, spontaneously hypertensive rats (male, 9-week-old, average body weight 280 g / animal) were prepared in four groups, six in each group.

The rats in each section were allowed to freely take each of the test foods I to IV for 2 weeks.

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

From the results in Table 5 above, the precipitated product of Example 2 (d)
The increase in blood pressure was hardly suppressed in the rats in the IV group to which the test diet containing the compound was administered, whereas the water-soluble hemicellulose of Reference Example (A) and the hemicellulose treated with the ultrafiltration membrane of Example 1 (C) Section II and II containing
In rats in the I group, the increase in blood pressure was suppressed,
It can be seen that the increase in blood pressure was significantly suppressed in the ward. And
This indicates that hemicellulose consisting of category A is effective in suppressing an increase in blood pressure. In addition, the water-soluble hemicellulose (a) used in the section II contains both the section A and the section B, and the section A contained therein is considered to act as an antihypertensive agent.

[Effect of the Invention] The antihypertensive and antihypertensive agent of the present invention exhibits an antihypertensive action and an antihypertensive action when administered in a small amount.

Further, according to the production method of the present invention, a high-purity antihypertensive or antihypertensive agent can be smoothly obtained with a simple operation.

Claims (4)

(57) [Claims] (1) The molar ratio of xylose: arabinose is 1: 0.
An antihypertensive or antihypertensive agent comprising a water-soluble hemicellulose having a sugar composition of 95 to 1.2. 2. A water-soluble hemicellulose is partially hydrolyzed and then treated with an ultrafiltration membrane to separate and collect a fraction having a sugar composition having a molar ratio of xylose: arabinose of 1: 0.95 to 1.2. The method for producing an antihypertensive / hypertensive agent according to claim 1. 3. After dissolving water-soluble hemicellulose in a buffer solution, adsorb it through an anion exchange resin, and then pass it through an eluent in which the concentration of sodium chloride is gradually increased from 0 mol to 0.4 mol. A xylose: arabinose molar ratio of 1: 0.95-1.2 eluted with 0.1-0.3 mole of eluent
The method for producing an antihypertensive / hypertensive agent according to claim 1, wherein the section having the sugar composition of (1) is recovered. 4. A 60% ethanol solution is added to water-soluble hemicellulose, and a precipitate formed therefrom is separated and removed from the supernatant to give a xylose: arabinose molar ratio of 1: 0.95 to 1.2.
The method for producing an antihypertensive / hypertensive agent according to claim 1, wherein the section having the sugar composition of (1) is recovered.