JPH09132595A - Immunological function promoter and its production - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new substance having cyclic phosphodiesterase IV activity- promotive action, thus useful in the medical and food industries, by adding an acid to an ethanol solution of zein as a kind of protein to partially decompose the zein followed by cooling and then filtration or centrifugation of the resultant precipitate. SOLUTION: An acid is added to an ethanol solution of zein as a kind of protein to partially decompose the zein, the resultant solution is cooled and the precipitate produced is recovered by filtration or centrifugal separation, thus obtaining the objective new immunostimulant having activity-promotive action on cyclic phosphodiesterase IV and hopeful of future wide applications in the medical and food industries. Specifically, this immunological function- promotive substance is obtained by the following process: corn gluten meal industrially separated from corn is subjected to extraction with a 70% ethanol solution, the resultant extract is dried into powdery zein which is then dissolved in ethanol, hydrochloric acid is added to the resultant solution to partially decompose the zein, the resultant solution is then cooled, the precipitate produced is recovered by filtration or centrifugal separation, and then decomposed by a protease.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD OF THE INVENTION

The present invention relates to an immune function-promoting substance and a method for producing the same, more specifically, cyclic phosphodiesterase IV.
The present invention relates to an immune function promoting substance, which is a zein decomposition product having an activity promoting action on (PDEIV), and a method for producing the same.

[0003]

[Prior art]

Cyclic phosphodiesterase (PDE) is
Cyclic adenosine-3 ', 5'-monophosphate (cAM
P) or cyclic guanosine-3 ', 5'-monophosphate (cGMP) is hydrolyzed to give 5'adenylic acid (5'-AMP) or 5'guanylic acid (5'GM, respectively).
It is an enzyme that converts to P), and currently there are 5 types (PDEI, PD
EII, PDEIII, PDEIV, PDEV) isozymes have been found, but newer isozymes are being discovered.

CAMP and cGMP which are substrates of PDE
Plays an important role as a second messenger for intracellular signal transduction, while PDE is an intracellular cA.
Intracellular cAMP, cG by hydrolysis of MP, cGMP
Since it is related to the regulation of MP concentration, it has important pharmacological significance.

It has been revealed that the expression of each PDE isozyme varies depending on the tissue or cell in the living body, and each PDE isozyme has a specific function. The search for a substance that promotes the activity of each PDE isozyme and suppresses it is pharmacological. It has become a big issue above.

For example, PDEI is mainly produced in tissue cells of the brain, heart, bronchus, etc., and therefore an inhibitor of PDEI activity is used as a smooth muscle relaxant.

Since PDEIII is mainly produced in tissue cells such as heart and bronchi, adipocytes, and platelets, the PDEIII activity inhibitor is effective as a cardiotonic agent and a platelet aggregation inhibitor.

[0009] Further, PDEIV is mainly produced in immunocompetent cells such as tissues such as brain and bronchus and lymphocytes, and promoting PDE activity in immunocompetent cells promotes the immune function of the living body. It is known.

[0010]

[Problems to be solved by the invention]

However, although there are some reports on the PDEIV inhibitor and its immunosuppressive effect, no report has been made on the activity-promoting substance.

Therefore, by finding a substance that promotes the activity of PDEIV, it is expected that the substance will be widely used in the fields of medicine and food as a novel substance that promotes immune function.

[0013]

[Means for Solving the Problems]

As a result of intensive studies to solve the above-mentioned problems, the present inventors have focused on corn protein as a material for a substance for suppressing or promoting the activity of PDE, and as a result of searching for its acid decomposition product and enzyme decomposition product. It was found that a specific fraction of acid lysate of protein zein has a PDEIV activity promoting action.

It has also been found that by further degrading a specific acid-degraded fraction with a proteolytic enzyme, its PDE activity promoting action is enhanced.

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

First, for cyclic phosphodiesterase IV obtained by adding an acid to an ethanol solution of protein zein to partially decompose it, and cooling the solution to recover a precipitate obtained by filtration or centrifugation. An immune function-promoting substance having an activity promoting action.

Secondly, an acid solution is added to an ethanol solution of protein zein to partially decompose it, and the precipitate obtained by cooling the solution is recovered by filtration or centrifugation, and then the recovered product is treated with a proteolytic enzyme. An immune function-promoting substance obtained by decomposing, which has an activity-promoting effect on cyclic phosphodiesterase IV.

Thirdly, an acid solution is added to an ethanol solution of protein zein to partially decompose it, and the precipitate obtained by cooling the solution is recovered by filtration or centrifugation to promote the activity against cyclic phosphodiesterase IV. A method for producing an immune function promoting substance, which comprises producing an immune function promoting substance having an action.

Fourthly, an acid solution is added to an ethanol solution of protein zein to partially decompose it, and the precipitate obtained by cooling the solution is collected by filtration or centrifugation, and then the recovered product is treated with a proteolytic enzyme. A method for producing an immune function-promoting substance, which comprises producing an immune function-promoting substance having an activity-promoting action on cyclic phosphodiesterase IV by decomposition.

The present invention will be described more specifically. The protein zein is dissolved in an ethanol solution having an ethanol concentration of 60 to 75% (V / V) so that the zein concentration becomes 2 to 8% (W / V). To do.

Hydrochloric acid was added to the solution so that the final concentration of hydrochloric acid was about 3%, and the temperature was kept at 45 to 60 ° C. in a sealed state.
Allow to react for 20-30 hours.

After completion of the reaction, the reaction solution is heated to 0 to 5 ° C. for 24 hours.
After standing for ~ 48 hours, the resulting precipitate is separated and collected by centrifugation or filtration, washed thoroughly with water, and then subjected to normal hot air drying and freeze drying to obtain a white powder of an acid partially decomposed product of zein. Obtain a function promoting substance.

Next, the acid partial decomposition product of zein obtained above was added in an amount of 8 to 12 times the amount of 6 to 8 M urea-0.5 to 2%.
Dissolve the solution in about ammonia water, apply this solution to a Biogel P-2 column equilibrated with 8-12 mM Tris-HCl buffer or the like (pH 8-9), develop with the same buffer, and prepare a solution from which urea has been removed. To do.

1/2 of the solid content of this preparation liquid
About 0.001 / 50 amount of proteolytic enzyme such as thermolysin or chymotrypsin is added and enzymatically digested for 2 to 4 hours at around the optimum temperature of each enzyme. Boil for ~ 40 minutes to inactivate its enzymatic activity.

Thereafter, the whole reaction solution is freeze-dried to obtain an immune function promoting substance which is an enzymatic digestion product of an acid partial decomposition product of zein obtained by enzymatically treating the acid-treated zein.

When the PDEIV activity-promoting activity of the enzyme-digested product was examined, it was confirmed that the activity-promoting activity was enhanced by 20 to 70% with respect to the acid partial decomposition product of zein, particularly at a low working concentration. , The enhancement effect is excellent.

[0028]

Example 1 (Preparation of AT-1, AT-2, AT-3)

Corn gluten meal industrially separated from corn was extracted with a 70% (V / V) ethanol solution, and dried zein was used as a sample.

A mixed solution of 2 liters of 99.5% ethanol and 1 liter of water was added to 100 g of the powdered zein, and the mixture was stirred and dissolved in a 5 liter beaker.

500 ml of constant boiling hydrochloric acid was added to the solution to prepare a stock solution for partial decomposition of acid.

The stock solution for partial decomposition of the acid was kept in a water bath so that the liquid temperature was 55 ° C., and the reaction was carried out for 24 hours while occasionally stirring.

After completion of the reaction, the reaction solution is put into ice water and
It was ice-cooled for 4 hours.

The precipitate produced by ice cooling was subjected to centrifugation of the reaction solution to separate it into a supernatant and a supernatant, and the precipitate was suspended in water and washed by centrifugation.

The water washing solution in this case was discarded.

The washed precipitate was freeze-dried and powdered to obtain 55 g of "AT-1".

The supernatant obtained by the above operation has a concentration of 40%.
Caustic soda was added to neutralize the mixture to pH 7.0, and the mixture was concentrated under reduced pressure on a rotary evaporator until ethanol was almost completely consumed.

The precipitate produced by the concentration under reduced pressure was centrifuged to collect the precipitate, and then the water suspension-centrifugation operation was repeated twice.

The cleaning liquid at this time was discarded.

The recovered precipitate was freeze-dried to give a powder,
20 g of a standard "AT-2" was obtained.

Glacial acetic acid was added to the supernatant obtained by the above-mentioned precipitation recovery to give a final concentration of 1% (W / W), and the resulting precipitate was collected by centrifugation and suspended in water. -The centrifugation operation was repeated twice, and the recovered precipitate was freeze-dried to "AT".
18 g of a standard product of "-3" was obtained.

[0042]

Example 2 (Preparation of AT-1, AT-2, AT-3)

The corn gluten meal was freeze-dried and n-
700 g of the degreased sample in a hexane / ethanol (1: 1) mixed solvent was placed in a 5 liter beaker,
Add 3000 ml of% (V / V) ethanol and adjust the liquid temperature to 6
Zein was extracted by keeping the mixture at 0 ° C. for 6 hours with occasional stirring.

The extract was left standing overnight at room temperature and then filtered to remove the extraction residue.

The filtrate was centrifuged (3500 rp).
m, 15 minutes) to remove the precipitate.

As a result, the solid content concentration was 7.5% (W / V).
2900 ml of clear solution of

To 2900 ml of this clear fluid, 360 ml of water
And 294 ml of 35% hydrochloric acid were added, and the reaction was carried out for 24 hours while occasionally stirring while maintaining the liquid temperature at 55 ° C.

After the reaction was completed, the solution was left standing at a liquid temperature of 4 ° C. for about 12 hours.

Since a precipitate was produced by this operation, the precipitate was recovered by performing the first centrifugation.

The recovered precipitate was further mixed with 1% aqueous ammonia 3
After suspending in 000 ml and stirring at 50 ° C. for 30 minutes, the remaining precipitate was collected by second centrifugation.

With respect to the recovered precipitate, the same operation was performed once more, and the finally recovered precipitate was freeze-dried to obtain 105 g of "AT-1".

2600 ml of the supernatant obtained by the first centrifugation operation was added with 5N caustic soda solution to neutralize the pH to 7.4, and then concentrated under reduced pressure by a rotary evaporator until most of ethanol was distilled off. did.

The concentrate was allowed to stand in ice water for 12 hours to complete the formation of a precipitate, which was then centrifuged to collect the precipitate.

The precipitate recovered was 1% ammonia solution 2
After suspending in liter and stirring at a temperature of 50 ° C. for 30 minutes,
The precipitate was recovered by centrifugation and the same operation was repeated once more to recover the precipitate.

The collected precipitate was freeze-dried to obtain "AT-
2 "66 g was obtained.

1800 ml of the supernatant of the first centrifugation during the preparation of "AT-2" was subjected to running water dialysis for 24 hours using a cellophane membrane, and the dialysate was concentrated by a rotary evaporator.

The concentrate was freeze-dried to obtain 10 g of "AT-3".

[0058]

Example 3 (Enzymatic treatment of AT-1 of Example 2)

1 g of AT-1 produced in Example 2 was placed in a 50 ml beaker, 20 ml of 1% ammonia water was added, and the mixture was dissolved by heating at 55 ° C.

After cooling, this solution was dialyzed against 2000 ml of deionized water using a cellulose membrane, and the dialysate was diluted to 5 times.
Transferred to a 0 ml beaker.

10 mg of chymotrypsin was added to this solution and digested for 2 hours at a temperature of 37 ° C. under the condition of pH 8.

After completion of the reaction, the product was treated in boiling water for 30 minutes and then freeze-dried to obtain 1 g of a standard product (AT-1-Chy).

Similarly, the enzyme was changed to pepsin, thermolysin, and subtilisin to adjust to optimum pH conditions (pH 2, pH 8, and pH 9, respectively) and digested at a temperature of 37 ° C. for 2 hours.

After completion of the digestion, the product was treated in boiling water for 30 minutes and then freeze-dried to prepare a standard product (AT-1-Pep, AT-1).
-Thm, AT-1-Sub) was obtained.

[0065]

Embodiment 4

20 g of AT-1 produced in Example 2 was placed in a 500 ml beaker, dissolved in 400 ml of 8M urea-1% ammonia water, and centrifuged (8000 rpm, 1).
0 minutes).

The obtained supernatant was treated with Cellulofine GCL.
Column packed with -25-m (diameter 10 cm x height 40
10 mM Tris-HCl buffer (pH 8.
The gel filtration fraction (AT-1U), which is a denatured protein from which urea was removed and which was developed in 5), was obtained in an amount of about 1000 ml.

In a 2000 ml beaker, add this AT-1
Take U, add 200 mg of thermolysin, add sodium hydroxide solution and keep the pH at 8 while keeping the temperature at 37.
Digestion was carried out at 0 ° C for 2 hours.

The digested liquid was treated in boiling water for 30 minutes and then freeze-dried to obtain 19 g of the standard product (AT-1U-Thm).
Obtained.

In the same manner as above, AT-1U was placed in another 2000 ml beaker, and chymotrypsin 200 mg was added.
Was added, and a sodium hydroxide solution was added to maintain the pH at 8, and digestion was performed at a temperature of 37 ° C. for 2 hours.

The digested liquid was treated in boiling water for 30 minutes and then freeze-dried to obtain 19 g of a standard product (AT-1U-Chy).

To 10 g of this AT-1U-Chy was added 500 ml of a 75% ethanol solution, the mixture was stirred and then filtered through a Nutsche, and the filter residue was freeze-dried to obtain a sample (AT-1U
-Chy (A)) was obtained.

Further, 250 ml of 75% ethanol solution was added to 5 g of AT-1U-Thm and stirred, and then filtered through Nutsche, and the filter residue was freeze-dried to obtain a sample (AT-1U
-Thm (A)) 1 g was obtained.

[0074]

[Test Example 1]

For "PDEI-IV", "AT-1,
In order to search the effect of "AT-2, AT-3" (acid treatment method zein 1, 2, 3), the test was carried out as follows.

First, "AT-1, AT-
2, AT-3 ”activity inhibition or promotion,
PDE isozymes have been described by Weislaar et al. (1986)
1), Silver et al. (1988), Reeves et al. (1987).

First, one medium-sized dog was anesthetized with ketalal, blood was sacrificed by arteriotomy, and then the chest was incised immediately to remove the heart.

Immediately ice-cool this heart, and
As a DE preparation amount, 10 to 15 g of left ventricular myocardium was cut out to obtain a PDE preparation material.

70 ml of an extraction buffer was added to this material, and the mixture was homogenized 4 times for 30 seconds using a homogenizer.

70 ml of extraction buffer used here
Is 10 mM Tris-HCl (pH 7.5), 2 m
M MgCl ₂ , 1 mM Dithiothreito
1, 1 μM (p-amino) PMSF, 100 μg /
ml water-soluble soybean Tripsin Inhibitor, 1
Prepared with 0 μg / ml leuptin.

The homogenite obtained was treated with 100,000 G
After ultracentrifugation at a temperature of 3 ° C. for 1 hour, the supernatant was filtered through glass wool and multi-layered gauze sequentially to obtain a liquid for column fractionation.

The solution for column fractionation was preliminarily equilibrated with an equilibration buffer (Pharmacia Q-S).
epharose fast flow bed, 30
ml).

The equilibration buffer used here was 70 m.
M sodium acetate (pH 6.5),
1 mM Dithiothreitol and 1 μM p
-Prepared by amino PMSF.

Then, after confirming that the absorbance of the effluent was lowered by washing the column with the above equilibration buffer, 420 ml of 70 mM to 1 M sodi was added.
Gradient elution of PDE isozymes with umacate.

The conditions for fractionation and elution were 1 ml / min and 7
This was a crude fractionation of PDEI-IV in ml / tube.

The fractionation result is illustrated in FIG.

Next, the PDE activities of PDEI to IV were measured by the method of Mukai et al. (1994).

The composition of the reaction solution for measuring PDE activity is shown in Table 1.

[0089]

[Table 1]

In the enzyme reaction, 0.5 ml of the reaction solution containing the substrate, the enzyme, and the test sample was reacted at a temperature of 30 ° C. for 10 minutes.

Then, the reaction was stopped by boiling for 5 minutes to inactivate the enzyme.

[0092] By this reaction, a ^[3 H] AMP produced in the reaction solution, after being converted to ^[3 H] adenosine by snake venom 5'-nucleotidase, and fractionated by cation exchange resins ^[3 The [H] concentration was measured by a liquid scintillation counter to test the effect of suppressing or promoting PDE activity.

In this test, the sample obtained in Example 1 was used as the sample, and the amount of the sample added to the enzyme reaction was 100 μg / ml.

The results are shown in Table 2.

The values in Table 2 are% of activity inhibition (-) or promotion (+) relative to the control (no addition of the sample).
The average value of the three tests was set to "0" in the suppression or promotion numerical value of 0 to 10%.

[0096]

[Table 2]

[0097]

[Test Example 2]

The following test was carried out to examine the PDEIV activity promoting effect of the enzyme-treated AT-1.

With respect to the PDE isozymes fractionated by the same method as in Test Example 1, the preparations of Examples 2 and 3 were examined for their PDE activity suppressing and promoting effects.

The results are shown in Table 3.

The values in Table 3 are the% of activity inhibition (-) or acceleration (+) relative to the control (no addition of the sample).
The average value of the three tests was set to "0" in the suppression or promotion numerical value of 0 to 10%.

[0102]

[Table 3]

[0103]

[Test Example 3]

The following test was conducted to examine the PDE activating effect of AT-1U-Thm and its gel-filtered fraction.

The AT-1U-Thm preparation of Example 4 was subjected to Q-Sepharose fa using an FPLC system.
20 mM to 1 M Amm using st flow column
Fractionation was performed by applying a linear gradient with an onium Bicarbononate.

As a result, as shown in FIG. 2, a, b, c,
Fraction could be fractionated in d.

AT of each of these fractions and Example 4
With respect to the -1U-Thm preparation, the inhibitory effect or the promoting effect on PDE was examined in the same manner as in Test Example 1.

In this test, "AT-1" of Example 2 and AT-1U-Thm of Example 4 were used as test samples.

Table 4 shows the results.

The values in Table 4 are% of activity inhibition (-) or promotion (+) relative to the control (no addition of the sample).
The average value of the three tests was set to "0" in the suppression or promotion numerical value of 0 to 10%.

[0111]

[Table 4]

[0112]

[Test Example 4]

From Test Examples 1 to 3, it was found that "AT-1" and its enzymatic degradation product had a PDEIV activity-promoting effect. Therefore, PDE fractionated in exactly the same manner as in Test Example 1
Using IV (ROLIPRAM IC50 = 0.65 μM), each preparation was tested for the amount of action and the effect of promoting activity.

In this test, the sample was
Using the preparations obtained in 3 and 4, the addition amount of the preparation to the enzyme reaction was 3 μg, 10 μg, 30 μg and 10 μg, respectively.
The accelerating effect without addition of the test sample was defined as 0%, and the accelerating effect was displayed.

The results are shown in Table 5.

The numerical values in Table 5 are% of activity acceleration (+) with respect to the control (no addition of the sample), and are the average values of two tests, and the acceleration numerical value of 0 to 10% is "0". "

[Table 5]

[0117]

【The invention's effect】

The immune function promoting substance according to the present invention is PDE
It has a substance that promotes IV activity and is expected to be widely used in the fields of medicine and food as a novel substance that promotes immune function.

[Brief description of the drawings]

FIG. 1 is a graph showing the results of fractionating PDE isozymes in Test Example 1.

FIG. 2 shows AT-1U-Th of Example 4 in Test Example 3.
Graph showing the result of fractionation of m standard

Claims (4)

[Claims] 1. An activity against cyclic phosphodiesterase IV, which is obtained by partially decomposing an ethanol solution of protein zein by adding an acid, and cooling the solution to recover a precipitate by filtration or centrifugation. An immune function promoting substance having a promoting action. 2. An ethanol solution of protein zein is partially decomposed by addition of an acid, and the precipitate obtained by cooling the solution is collected by filtration or centrifugation, and then the recovered product is decomposed by a proteolytic enzyme. An immune function-promoting substance thus obtained, which has an activity-promoting effect on cyclic phosphodiesterase IV. 3. A protein zein ethanol solution is partially decomposed by addition of an acid, and the precipitate obtained by cooling the solution is collected by filtration or centrifugation to have an activity promoting action on cyclic phosphodiesterase IV. A method for producing an immune function-promoting substance, comprising producing an immune function-promoting substance having the same. 4. An ethanol solution of protein zein is partially decomposed by addition of an acid, and the precipitate obtained by cooling the solution is collected by filtration or centrifugation, and then the collected product is decomposed by a proteolytic enzyme. Cyclic phosphodiesterase
A method for producing an immune function promoting substance, which comprises producing an immune function promoting substance having an activity promoting action on IV.