JPH08245694A - Vasodepressor substance and zein capable of being easily degraded with protease and used as its production intermediate and their production - Google Patents

Abstract

PURPOSE: To obtain a vasodepressor substance having an angiotensin-converting enzyme-inhibiting effect and useful for foods, medicines such as a hypertension- preventing and treating medicine, etc., by dissolving and denaturating zein with a protein-denaturating agent, removing the protein-denaturating agent, and subsequently degrading the treated zein with a protease. CONSTITUTION: Zein contained in corn is isolated by a method comprising solvent extraction, defatting and drying processes, dissolved and denaturated in a 8M urea-1% ammonia solution containing a protein-denaturating agent such as urea, and subsequently subjected to a gel filtration process using a gel column balanced with a 10mM tris hydrochloride buffer solution (pH: 8.5) to separate off the protein-denaturating agent. The left white emulsion-like denaturated zein is collected, controlled with a 0.5N sodium hydroxide aqueous solution to a pH of approximately 8.0, degraded with a protease such as thermolysin for approximately 2hrs, and subsequently boiled to thermally deactivate the enzyme, thus affording the vasodepressor substance having an angiotensin-converting enzyme (ACE)-inhibiting effect and useful for foods, health foods, medicines used for preventing and treating hypertension, etc.

Description

Detailed Description of the Invention

[0001]

[Industrial applications]

The present invention relates to an antihypertensive substance, a protease-degradable zein which is an intermediate for producing the same, and a process for producing them.

The blood pressure lowering substance according to the present invention can be used as a medicine, food or health food for the prevention and treatment of hypertension.

[0004]

[Prior art]

There are several mechanisms for controlling blood pressure.

In the renin-angiotensin system, angiotensin nogen secreted from the liver is converted into angiotensin-I by renin existing in the kidney, and angiotensin-I is angiotensin-converting enzyme (hereinafter abbreviated as ACE) in the blood. I
Converted to I.

Angiotensin-II is a potent pressor peptide, which contracts vascular smooth muscle and raises blood pressure.

[0008] ACE also acts on the kallikrein-quinine system and decomposes the antihypertensive bradykinin to increase blood pressure.

By inhibiting this ACE, the rise in blood pressure can be suppressed.

As the ACE inhibitor, captopril and the like have been put into practical use as pharmaceuticals.

Further, many drugs as ACE inhibitors,
Highly purified peptides and foods have been reported.

[0012] However, highly purified peptides are not practical as foods, and there are many doubtful ones such as those merely reported as foods or health foods, because their effects are weak or not long-lasting.

Most of the conventional ACE-inhibiting peptides are those obtained by highly purifying proteolytic products and those synthesized from amino acids.

From the viewpoint of cost, these were not practical as foods.

Further, some blood pressure-lowering substances have been obtained from soybean, wheat and the like, and they have been reported as pharmaceuticals and foods, but none showing sufficient effects have been found.

[0016] For example, Japanese Patent Application Laid-Open No. 2-240028 describes a thermolysin hydrolyzate of zein as a blood pressure lowering agent.

[0017]

[Problems to be Solved by the Invention]

However, the above-mentioned Japanese Patent Laid-Open No. 2-2400
The antihypertensive agent described in Japanese Patent No. 28 is not suitable for industrial production because zein does not dissolve in water and it takes time to decompose zein.

Therefore, an object of the present invention is to provide pharmaceuticals, foods,
Another object is to provide a blood pressure-lowering substance that is safe and practical as a health food, has an ACE inhibitory effect, and is suitable for industrial production.

[0020]

[Means for Solving the Problems]

As a result of various investigations to solve these problems, the present inventors have found that water-insoluble zein, which is a protein of corn, is used in the above-mentioned JP-A-2-240.
As described in Japanese Patent Application No. 028, the solution is not decomposed directly by an enzyme in a suspended state, but is dissolved in a solution of a protein denaturant such as urea containing ammonia and the like, and further treated with gel filtration column or the like to give zein. After separating a protein denaturing agent such as urea and making zein susceptible to enzymatic degradation, it is effective as a blood pressure lowering agent with a significant ACE inhibitory effect on what is decomposed with a proteolytic enzyme such as thermolysin. The inventors have found that they can be used for the treatment and prevention of hypertension and completed the present invention.

That is, the means for solving the problems of the present invention are as follows.

First, a hypotensive substance obtained by dissolving and denaturing zein with a protein denaturing agent, removing the denaturing agent, and decomposing it with a protease.

Secondly, a hypotensive substance obtained by dissolving and denaturing zein with urea, removing urea by gel filtration, and decomposing it with thermolysin.

Thirdly, a protease-degradable zein obtained by dissolving and denaturing zein with a protein denaturing agent and then removing the denaturing agent.

Fourth, a protease-degradable zein obtained by dissolving and denaturing zein with urea and then removing the urea by gel filtration.

Fifth, a method for producing a blood pressure-lowering substance produced by dissolving and denaturing zein with a protein denaturing agent, adding a property of easily undergoing enzymatic decomposition, removing the denaturing agent, and decomposing it with a protease. .

Sixth, a method for producing a blood pressure lowering substance produced by dissolving and denaturing zein with urea, adding a property of easily undergoing enzymatic decomposition, removing urea by gel filtration and decomposing with thermolysin.

Seventh, a method for producing a protease-degradable zein which is produced by dissolving and denaturing zein with a protein denaturing agent to add a property of easily undergoing enzymatic decomposition and then removing the denaturing agent.

Eighth, a method for producing a protease-degradable zein produced by dissolving and denaturing zein with urea, adding a property of easily undergoing enzymatic decomposition, and then removing urea by gel filtration.

According to the present invention, by dissolving and denaturing zein with a protein denaturing agent, the intramolecular bond of zein becomes gradual and the zein becomes susceptible to enzymatic action.

Then, by rapidly decomposing a substance that is susceptible to the enzyme action with a protease, A
An enzymatic decomposition product of zein having CE inhibitory activity can be obtained in a short time in a high yield.

As the protein denaturant used in the present invention, urea or guanidine hydrochloride can be used.

When dissolving zein, ammonia or caustic soda can be used.

The removal of the protein denaturing agent is not particularly limited, but the kind of gel may be optional, and Seph may be used.
A gel such as adex that can be molecular sieve can be used.

The buffer is not particularly limited, and zein can be separated by flowing a buffer according to the type of gel.

The pH at the time of separation can be 7.0 to 10.0, but it is preferably around 8.5. When the pH is less than 7.0, zein is precipitated and the pH is 10.0. If it exceeds, the separation of zein from impurities such as pigments becomes worse.

The conditions such as pH when decomposing with an enzyme such as thermolysin can be selected according to the enzyme used.

Upon decomposition, the pH is kept constant by using, for example, 0.5N sodium hydroxide.

[0040]

Embodiment 1

Zein contained in corn was prepared by solvent extraction, degreasing and drying, and dissolved in 8M urea-1% ammonia solution to denature zein.

Then, 10 mM Tris-HCl buffer (p
H8.5) equilibrated gel column (trade name = Bio-
The denaturant was separated and removed using Gel P-2).

The separated white milky liquid modified zein was collected, and the zein was decomposed with thermolysin for about 2 hours using 0.5N sodium hydroxide so as to have a pH of about 8.0, and heat-denatured by boiling. The enzyme was deactivated and the enzyme activity was stopped.

Any enzyme can be used for the decomposition, but when thermolysin is used, a decomposition product having a relatively high inhibitory activity can be obtained.

When stopping the reaction of the enzyme, an enzyme inhibitor or the like may be added in addition to boiling.

The obtained decomposition product was modified by Yamamoto et al. Of the Lieberman method (Journal of the Japanese Society of Chest Diseases 18
(5), P297-303, 1980), ACE
IC50 (concentration of 50% inhibition, hereinafter abbreviated as IC50), which is an index of inhibitory activity, was measured to be 40 μg /
It was less than or equal to ml, and the decomposed product obtained here had sufficient activity even if decomposed within 3 hours.

However, the inhibitory activity did not change even if the decomposition time was lengthened.

In the following examples, the IC50 is Lieb
It was measured by a modified method of Yamamoto et al. of the erman method.

Here, without modifying the zein,
In the direct decomposition method using thermolysin in a state of being suspended in water, decomposition took about 24 hours and the yield was remarkably low.

[0050]

Example 2

500 mg of zein prepared in the same manner as in Example 1 was dissolved in 10 ml of 8M urea-1% ammonia solution to denature the protein.

Next, 10 mM Tris-HCl buffer (pH
Bio-Gel P-2 (f equilibrated in 8.5)
urea) was separated and removed from the zein using an (ine) column (φ3 cm × 40 cm).

The isolated zein was obtained as a white cloudy opalescent liquid.

To this, 2.5 ml of a 1 mg / ml thermolysin (manufactured by Wako Pure Chemical Industries) solution was added, and the pH was adjusted to 40 ° C.
Decomposition was performed at 8.0 for 2 hours.

After the completion of decomposition, the enzymatic reaction was stopped by treating in boiling water for 30 minutes and freeze-dried under vacuum to obtain 310 mg of powder.

IC50 (μg) for ACE of dry powder
/ Ml) was measured by a modified method of the Lieberman method by Yamamoto et al. In the same manner as in Example 1 and found to be 16.6 μm.
It was g / ml and had sufficient activity.

[0057]

Example 3

Separation and removal of urea from the urea-modified zein solution by gel filtration was examined as follows.

10 g of zein was added to 400 ml of 8M urea-
After dissolving in 1% ammonia solution to denature the protein, 1
Bio-Gel P-2 (fine) column (φ10c) equilibrated with 0 mM Tris-HCl buffer (pH 8.5).
The urea was separated using m × 40 cm).

Then, for each of the obtained fractions, the absorbance derived from zein (OD = 280 nm) and urea were measured.

A Hitachi spectrophotometer (U-20
00), and urea nitrogen-Test Wako (manufactured by Wako Pure Chemical Industries, Ltd.) was used for urea measurement.

The measurement results are shown in FIG.

From FIG. 1, it can be confirmed that urea and zein are clearly separated.

The zein fraction was collected, 100 mg of thermolysin (manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved in 100 ml of water, and the mixture was decomposed at 40 ° C. for 2 hours.

Then, the enzyme reaction was stopped by heating in boiling water for 30 minutes, and freeze-drying was carried out to obtain 6.93 g of freeze-dried powder.

[0066]

Embodiment 4

27.5 g of zein was added to 550 ml of 8M
After dissolving in urea-1% ammonia solution and stirring overnight,
The precipitate was removed by centrifugation at 8000 rpm for 20 minutes, and urea was separated and removed using a gel filtration column.

Column separation was performed using Bio-Gel P-2.
(Fine) was packed in a column of φ120 mm × 1000 mm to a height of 400 mm and sufficiently equilibrated with 10 mM Tris-HCl buffer (pH 8.5).

The cloudy portion separated by the column was collected to obtain about 800 ml of easily degradable zein.

Thermolysin (manufactured by Wako Pure Chemical Industries) 25
0 mg was dissolved in 250 ml of water, added, and decomposed at 37 ° C. for a certain time (1, 2, 3 or 6 hours).

The decomposition was carried out while adjusting the pH to 8.0 using 0.5N sodium hydroxide.

After completion of the decomposition, the product was treated in boiling water for 30 minutes and freeze-dried to obtain decomposed products at each decomposition time.

The IC50 for ACE was measured for each of the obtained decomposition products at each decomposition time.

The results were as follows.

The IC50 for a decomposition time of 1 hour is 3
It was 1.6 μg / ml. I with a decomposition time of 2 hours
C50 was 16.1 μg / ml. The IC50 of the one having a decomposition time of 3 hours was 18.3 μg / ml.
The IC50 for a decomposition time of 6 hours is 23.9 μg / m
It was l.

When the relationship between the enzymatic decomposition time in zein after gel separation and the IC50 was examined, it was found that the IC50 was lowest and the ACE inhibitory activity was high when the enzymatic decomposition time was 2 hours.

[0077]

Example 5

Regarding α-zein, which constitutes the majority of corn protein zein, the peptides in the enzymatic degradation products were fractionated to determine the amino acid sequence and the relationship with the ACE inhibitory activity was investigated as shown below. Carried out.

100 mg of crude α-zein obtained by extraction with 70% ethanol was dissolved in 4M urea-1% ammonia solution at 50 ° C. and allowed to stand, and then urea was separated and removed by gel filtration in the same manner as in Example 4. Then, modified zein was obtained.

Thermolysin in an amount of 1/100 to zein was added to the modified zein, and the mixture was decomposed for 3 hours while adjusting the pH to 8.0 at 37 ° C., and freeze-dried under vacuum.

After freeze-drying under vacuum, the decomposed product was dissolved in 100% ethanol and centrifuged to separate into a supernatant and a precipitate.

The supernatant was concentrated to give an F-1 fraction, the precipitate was dissolved in 75% ethanol and centrifuged, and the supernatant was concentrated to give an F-2 fraction.

The IC50 of the F-1 and F-2 fractions were 22 μg / ml and 43 μg / ml, respectively.

The fractionation of the peptide was carried out by reverse phase-HPLC Long C18 C manufactured by JASCO Corporation in a 0.1% TFA solution.
It was performed using the column.

The results are shown in FIG.

Further, for each of the obtained fractions, the relative activity of ACE inhibition was determined and the amino acid sequence was determined.

The relative activity of ACE inhibition of each fraction and the IC-50 of the corresponding peptide (see also known literature) are shown in Table 1 [F-1 fraction (100% ethanol)],
It is shown in Table 2 [F-2 fraction (75% ethanol)].

[0088]

[Table 1]

[0089]

[Table 2]

When these results are examined, many of them having a low IC50 are seen, and it is considered that the overall ACE inhibitory activity is increased.

[0091]

Example 6

5 g of zein was added to 100 ml of 8M urea-
It was dissolved in a 1% ammonia solution, centrifuged at 6000 rpm for 15 minutes to remove the precipitate, and the obtained supernatant was separated and removed by a gel filtration column.

Column separation was carried out by Bio-Gel P-2 column (φ30, equilibrated with 10 mM Tris-HCl buffer).
mm × 400 mm) to separate and remove urea from zein.

To the separated zein solution, 50 mg of thermolysin (manufactured by Wako Pure Chemical Industries) dissolved in water was added.
Decomposition was carried out at 0 ° C for 2 hours.

Similarly, Denateam (Nagase Seikagaku Corporation)
Subtilisin (Boehringe)
r Mannheim), Papain (Boehrin)
Germann Mannheim) and Protease A (Amano Pharmaceutical Co., Ltd.) were dissolved in water at a concentration of 50 mg, and each of them was decomposed at 40 ° C. for 2 hours.

The decomposition was carried out while adjusting the pH using a 0.5N sodium hydroxide solution.

At this time, the required amount of 0.5N sodium hydroxide solution required for neutralization is shown in FIG.

After the decomposition, it was treated in boiling water for 30 minutes and freeze-dried to obtain decomposed products at each decomposition time.

Then, the IC50 for ACE was measured for each of the obtained decomposition products at each decomposition time.

The results are as follows.

When thermolysin was used, the pH upon decomposition was 8.0 and the IC50 was 31.2 μg / ml.

When papain was used, the pH upon decomposition was 7.5 and the IC50 was 135.0 μg / ml.

When Denazyme was used, the pH during decomposition
Was 8.0 and the IC50 was 450.7 μg / ml.

When Subtilisin was used, the pH at the time of decomposition was 8.5 and the IC50 was 332.3 μg.
/ Ml.

When Protease A was used, the pH at the time of decomposition was 8.0 and the IC50 was 206.7 μg / ml.
Met.

Considering the above results, activity was found in all the enzymes, but thermolysin was the best.

[0107]

[Test example]

The bioactivity of the hydrolyzate of the present invention against pathological animals was tested as follows.

Zein degradation products (1, 2, 3) obtained in Example 4
Or 6-hour degradation) was orally administered once to stroke-prone spontaneously hypertensive rats (SHRSP), and then the blood pressure of the rats was changed over time.
Method (Muromachi Kikai KK MK-1030 type) was used for each measurement.

The results are shown in FIG. 4, which shows the actual measurement data as the average value of 5 animals, and in FIG. 5, the blood pressure fluctuation value from the state before administration is shown in comparison with the control.

Considering these results, the blood pressure of the water-supplied (control) was almost constant, but the blood pressure of the zein-decomposed product was 20 mmHg at the maximum as compared with the water-supplemented one. Showed a descent.

The zein degradation product obtained in Example 4 was 2
It can be confirmed that there is a blood pressure lowering effect even after 4 hours, and the effect continues for a long time.

Therefore, according to the present invention, the enzyme treatment time in the production process can be greatly shortened, which is suitable for industrial production, the zein degradation product thus obtained has a high inhibitory activity and a long blood pressure lowering action. Lasts for hours.

[0114]

【The invention's effect】

According to the present invention, it is safe and practical as a pharmaceutical, food or health food and has an ACE inhibitory effect,
A blood pressure lowering substance suitable for industrial production can be provided.

[Brief description of drawings]

FIG. 1 shows the results of gel filtration separation.

FIG. 2 is a diagram showing the results of separation by HPLC.

FIG. 3 is a diagram showing the required amount of sodium hydroxide solution required for neutralization during hydrolysis of urea-removed zein solution by various proteases.

FIG. 4 is a graph showing the effect of zein decomposition products on blood pressure.

FIG. 5 is a graph showing the effect of zein degradation product on blood pressure as a blood pressure fluctuation value for control.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display area A61K 38/00 ABU A61K 37/18 ABU

Claims (8)

[Claims] 1. A blood pressure-lowering substance obtained by dissolving and denaturing zein with a protein denaturing agent, removing the denaturing agent, and decomposing it with a protease. 2. A hypotensive substance obtained by dissolving and denaturing zein with urea, removing urea by gel filtration, and decomposing it with thermolysin. 3. A protease-degradable zein obtained by dissolving and denaturing zein with a protein denaturing agent and then removing the denaturing agent. 4. A protease-degradable zein obtained by dissolving and denaturing zein with urea and then removing the urea by gel filtration. 5. Zein is dissolved and denatured with a protein denaturant,
A method for producing a hypotensive substance, which is produced by adding a property of easily undergoing enzymatic decomposition, then removing the denaturing agent, and decomposing with a protease. 6. A method for producing a blood pressure-lowering substance produced by dissolving and denaturing zein with urea, adding a property of easily undergoing enzymatic decomposition, removing urea by gel filtration, and decomposing with thermolysin. 7. Zein is dissolved and denatured with a protein denaturant,
A method for producing a protease-degradable zein, which is produced by adding a property of easily undergoing enzymatic decomposition and then removing the denaturing agent. 8. A method for producing a protease-degradable zein, which is produced by dissolving and denaturing zein with urea, adding a property of easily undergoing enzymatic decomposition, and then removing urea by gel filtration.