JPH0725793B2 - Angiotensin converting enzyme inhibitor - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to angiotensin I and angiotensin II.
The present invention relates to an angiotensin-converting enzyme inhibitor that inhibits the activity of angiotensin-converting enzyme that is converted into.

[Prior Art] It has been conventionally known that angiotensin-converting enzyme has an action of converting angiotensin I into angiotensin II.

And thus, angiotensin II converted from angiotensin I by angiotensin converting enzyme in this way
Is said to have the effect of increasing blood pressure.

Therefore, by inhibiting the activity of angiotensin-converting enzyme that converts angiotensin I into angiotensin II, it is possible to suppress an increase in blood pressure, and conventionally, there are various substances that inhibit the activity of angiotensin-converting enzyme as described above. Various studies have been carried out, and various angiotensin converting enzyme inhibitors have been developed.

And, as a substance that inhibits the activity of the angiotensin converting enzyme as described above, conventionally, for example, D-
In addition to synthetic substances such as 2-methyl-3-mercaptopropanoyl-L-proline, casein is disclosed in JP-A-62-270533, JP-A-64-5497 and JP-A-64-83096. Various peptides obtained by decomposing the above are disclosed.

However, the angiotensin-converting enzyme inhibitor composed of the above-mentioned compound generally has a high inhibitory activity of angiotensin-converting enzyme, but since it is a compound, it often has some problems in terms of safety such as side effects. It was

On the other hand, in the case of an angiotensin converting enzyme inhibitor obtained by decomposing casein etc., its inhibitory activity is generally lower than that of the above-mentioned synthetic product, but its raw material is a natural product, especially because it is derived from food. Generally, the toxicity was low and the safety was high.

Therefore, further development of natural products, particularly food-derived angiotensin-converting enzyme inhibitors as described above, has been desired.

[Problems to be Solved by the Invention] The present invention has been made in view of the above circumstances, and effectively inhibits the activity of an angiotensin-converting enzyme that converts angiotensin I into angiotensin II, thereby increasing blood pressure. It is an object of the present invention to provide an angiotensin converting enzyme inhibitory substance that can be suppressed, is highly safe to the human body, is administered as a food, etc., and can suppress an increase in blood pressure with a mild action.

[Means for Solving the Problems] In the present invention, as an angiotensin-converting enzyme inhibitor that inhibits the activity of angiotensin-converting enzyme that converts angiotensin I into angiotensin II,
The peptide of the structural formula [1] shown below or a salt thereof was used.

[1] Asp-Gln-Thr-Pro-Arg-Val-Phe Here, as the salt form in the above-mentioned peptide, there are inorganic salts such as hydrochloride, hydrobromide, hydroiodide, sulfate and phosphate. Acid salt, acetate, trifluoroacetate, citrate,
Examples of the organic acid salt include maleate, fumarate, tartrate, lactate, methanesulfonate and P-toluenesulfonate.

Further, the amino acids constituting the above peptides are preferably L-forms in that they naturally occur.

Here, the peptide represented by the structural formula [1] above can be produced by degrading isolated soybean protein isolated from soybean with pepsin.

The peptide represented by the structural formula [1] and a salt thereof have an action of inhibiting the activity of angiotensin-converting enzyme that converts angiotensin I into angiotensin II, and from soybeans used as food as described above. The obtained isolated soybean protein is derived from a food product that can be produced by decomposing it with pepsin, and is highly safe.

[Examples] Examples of the present invention will be specifically described below.

First, the case of producing the peptide having the structural formula shown in [1] above will be described.

In this example, 3.0 g of separated soybean protein (Fuji Pro-R manufactured by Fuji Oil Co., Ltd.) separated from soybean was dissolved in water, and acetic acid was added to the solution to adjust the pH to 3.2. The resulting solution was adjusted to pH 2.0 by adding hydrochloric acid to make 60 ml of solution.

Then, pepsin was added to this solution at 1/50 of the soybean protein isolate.
An amount of 0, that is, 6 mg was added, and this was reacted at 37 ° C. for 10 hours, and the above isolated soybean protein was enzymatically decomposed by pepsin.

Then, this was heated at 100 ° C for 15 minutes to inactivate the above pepsin, and then this was centrifuged at 10000 rpm for 2 minutes.
Centrifugation was carried out for a minute to separate into a precipitate fraction and a solution fraction, the precipitate fraction was removed, and only the solution fraction was taken out.

Then, the solution fraction thus taken out was fractionated using molecular sieve chromatography (Pharmacia Sephadex G-25).

Here, in performing the fractionation by the above-mentioned molecular sieve chromatography, a column volume of 200 ml is used, a 0.05M acetic acid solution is used as a developing solution, and the flow rate is 4 ml / hr. In each fraction
I tried to collect 3 ml each.

Next, the amount of protein and the angiotensin converting enzyme inhibitory rate of each of the fractions thus collected were measured.

Here, in measuring the protein amount, in this example, the known TNBS method was used.

That is, in this example, 0.10% TNBS (2,4,6-
Trinitrobenzene sulfonic acid) solution 0.5 ml, 0.01M
0.5 ml of sodium sulfite solution of, and 2.0 ml of 0.15 M borate buffer, 0.5 ml of appropriately diluted sample in each of the above fractions so as to fall within the measurement range of absorbance, After reacting these at 37 ° C. for 60 minutes, the absorption of light at 420 nm was measured by a spectrophotometer, and the result is shown by the solid line in FIG. The larger the absorption of light at 420 nm measured by the spectrophotometer, the larger the number of peptides in the sample.

Further, in measuring the angiotensin-converting enzyme inhibition rate in each fraction collected as described above,
In this example, the angiotensin-converting enzyme inhibitory rate was measured by a known method for measuring angiotensin-converting enzyme inhibitory activity (Cushman-Cheung method).

Here, in this example, 10 g of rabbit rung acetone powder (manufactured by Sigma) was dissolved in 100 ml of 50 mM borate buffer (pH 8.3), and after centrifugation at 40,000 g for 40 minutes, Supernatant with 5OmM potassium phosphate buffer 5
Diluted by a factor of two to prepare an angiotensin converting enzyme solution.

On the other hand, each of the above fractions was freeze-dried and then dissolved in 0.6 ml of 50 mM potassium phosphate buffer, and 50 μm of each sample was collected from this solution.

Then, for each sample of 50μ collected in this way, the substrate Bz-Gly-His-Leu (manufactured by Peptide Institute), a mixed solution of potassium phosphate buffer and sodium chloride, as described above. The prepared angiotensin converting enzyme solution (50 μ) was added to make 250 μ. At this time, the final concentration of the substrate in the above solution was 2.5 mM, the final concentration of the calcium phosphate buffer was 100 mM, and the final concentration of sodium chloride was 300 mM.

Then, each of the mixed solutions thus prepared was reacted at 37 ° C. for 30 minutes, 250 μ of 1N hydrochloric acid was added to stop the reaction, and then 1.5 ml of ethyl acetate was added to the mixture to give 10
Stir for 2 seconds, then centrifuge at 35000 rpm for 2 minutes,
1 ml of the ethyl acetate layer was collected.

Then, the thus collected material was dried to dryness with nivaporate, and then dissolved in 1 ml of distilled water,
The absorption of the extracted hybric acid (absorbance at 228 nm) was measured, and the angiotensin-converting enzyme inhibition rate of the sample in each fraction was determined by the above formula [1].

(However, OD _s is the absorbance when the sample is added and measured as described above. OD _sb is the absorbance when 250 μl of 1N hydrochloric acid is added before the reaction of the above mixed solution. OD _c Is the absorbance when measured without adding a sample to the above mixed solution, and OD _cb is the absorbance when measured with 250 μl of 1N hydrochloric acid added to the above mixed solution without adding a sample.) The angiotensin-converting enzyme inhibitory rate (ACE inhibitory rate) in the samples of the respective fractions measured as described above is shown by the broken line in FIG.

As a result, as shown in the figure, the angiotensin-converting enzyme inhibition rate was high in the fraction numbers 44 to 60.

Therefore, in this example, the fraction number
The portion of 44 to 60, that is, the one having a molecular weight of about 2000 to 200 was collected.

Then, the thus separated fraction is freeze-dried, and then the freeze-dried fraction is further fractionated by using molecular sieve chromatography (Pharmacia Sephadex G-10). I did it.

Here, when fractionating by this molecular sieve chromatography, a column volume of 150 ml was used, and a 0.05M acetic acid solution was used as a developing solution, and the flow rate was 3 ml / hr. Then, 1 ml of each fraction was collected.

Then, with respect to the fractions thus separated into fractions, the protein amount and the angiotensin converting enzyme inhibitory rate in each fraction were measured in the same manner as in the above case.

And, while the absorption result of light at 420 nm in each fraction measured by the spectrophotometer is shown by a solid line in FIG. 2, the angiotensin converting enzyme inhibition rate (ACE inhibition rate) in each sample collected from each fraction is the same. It is indicated by a broken line in the figure.

As a result, as shown in FIG. 2, the fraction number is 10
In the case of 5-130, the angiotensin-converting enzyme inhibitory rate was high with respect to the number of peptides.

Therefore, in this example, the fraction number
Those having a molecular weight of 105 to 130, that is, those having a molecular weight of about 1000 to 400 were collected.

Then, the thus collected fractions were fractionated by cation exchange separation.

Here, in the cation exchange separation, an FPLC unit manufactured by Pharmacia was used, a cation exchange resin Mono S HR5 / 5 (trade name) was used as a column, and 0.05 M of a developing solution was used. While using acetic acid-ammonia buffer (pH 7.5), 0.5M as a gradient
Sodium chloride solution is used, and the amount of the sodium chloride solution added is set to 0 for 5 minutes from the beginning, and is increased to 0 to 100% in 5 to 25 minutes so that the flow rate is 1 ml / min. , 0.5 ml of each fraction was collected.

Then, the amount of protein and the angiotensin converting enzyme inhibitory rate of each of the fractions fractionated as described above were measured.

Here, in measuring the angiotensin-converting enzyme inhibition rate in each fraction, after freeze-drying each fraction, 50 mM each of these
Dissolve it in 1 ml of potassium phosphate buffer, and otherwise measure the angiotensin-converting enzyme inhibitory rate (ACE inhibitory rate) of each sample in the same manner as in the angiotensin-converting enzyme inhibitory activity measurement method described above. The results are shown in FIG. 3 (A).

On the other hand, the amount of protein in the sample in each fraction was measured by a known ultraviolet absorption method at 280 nm.
The absorption of light was measured and the results are shown in FIG. 3 (B). Even in this case, the larger the absorption of light at 280 nm, the larger the amount of protein in the sample.

As a result, as shown in FIG. 3 (A), the angiotensin converting enzyme inhibitory rate was low in the flow-through fraction at the time when the 0.5 M sodium chloride solution was not added.

Then, in this example, fractions with a fraction number of 28,29 in which the angiotensin converting enzyme inhibitory rate was high in FIG. 3 (A) were fractionated. Then
The collected fractions with fraction numbers 28 and 29 were lyophilized, and this sample was dissolved in 40 μl of distilled water and then subjected to high performance liquid chromatography (HPLC manufactured by Hitachi, Ltd.).
Further fractionation was performed using L-4200).

Here, in the high performance liquid chromatography fractionation, a solution A containing 0.05% tetrahydrofurfuryl acrylate (TFA) and 5% acetanilide, and 0.05% TFA and 100% acetanilide were added. B
Solution, by adding only the above solution A for 5 minutes from the beginning, and decreasing the above solution A to 100 to 0% and increasing the solution B to 0 to 100% for 5 to 35 minutes. And fractionated.

Then, the thus-fractionated product was measured for its protein content by the aforementioned ultraviolet absorption method.

As a result, light absorption results as shown in FIG. 4 were obtained for light having a wavelength of 280 nm in the samples using fraction numbers 28 and 29 as samples.

Then, in the figure, the portion (a) which absorbs light to some extent and contains an appropriate amount of protein was fractionated, and the portion (a) thus fractionated was freeze-dried. rear,
This was dissolved in 0.4 ml of distilled water, 20 μm of this solution was collected, and a sample of 50 μm was prepared by adding 30 μm of 50 mM potassium phosphate buffer, and the angiotensin-converting enzyme inhibitory activity measurement method described above was used. Similarly, when the angiotensin-converting enzyme inhibition rate was measured, the angiotensin-converting enzyme inhibition rate in this sample was 39.9%.
And showed a high value.

Then, the portion (a) shown in FIG. 4 is separated and freeze-dried to completely remove the solvent.
In the same manner as in the above case, the purification was performed again by the above high performance liquid chromatography, and the amount of the protein was measured by the ultraviolet absorption method.

As a result, in the product purified from the portion (a) shown in FIG. 4, the absorption result of light having three peaks as shown in FIG. 6 was obtained for the light of 280 nm.

Then, in the figure, when the angiotensin-converting enzyme inhibition rate was measured for each of the first, second, and third mountain portions (a ₁ ), (a ₂ ), and (a ₃ ) as described above, The angiotensin-converting enzyme inhibition rate of the part (a ₁ ) is almost 0%, the angiotensin-converting enzyme inhibition rate of the part (a ₂ ) shows a high value of 33.1%, and the angiotensin-converting enzyme part of the (a ₃ ) shows a high value. The inhibition rate was 16.1%.

Therefore, the portion (a ₂ ) in which the angiotensin converting enzyme inhibition rate was high as described above was fractionated from the portion (a).

Then, it is shown in FIG. 5 which was taken out as described above (a ₂ ).
The structure of the peptide contained in the above portion was identified by an amino acid sequencer (Model 477A manufactured by Applied Biosystems).

As a result, it was found that the portion (a ₂ ) shown in FIG. 5 was a peptide represented by the following structural formula [1].

[1] Asp-Gln-Thr-Pro-Arg-Val-Phe And the above-obtained peptide of structural formula [1] is assayed for the angiotensin-converting enzyme inhibitory activity (Cushman-Cheung method). Accordingly, where the angiotensin converting enzyme inhibition rate was determined concentration ID ₅₀ of the required peptide to be 50%, in the peptide shown in the above formula [1], the ID ₅₀ was 42MyuM.

Moreover, when it was confirmed whether the peptide represented by the structural formula [1] was derived from the isolated soybean protein used as the starting material, it was found that the amino acid sequence represented by the structural formula [1] was soybean. The protein fraction of protein is 11sA ₅ A ₄ B
₃ subunits are present in the precursor sequence,
It was confirmed that the peptide represented by the structural formula [1] was derived from the isolated soybean protein used as the starting material.

Therefore, the peptide represented by the structural formula [1] can effectively inhibit the activity of angiotensin-converting enzyme that converts angiotensin I into angiotensin II as described above, and can be administered as a food or the like. It is expected that the increase in blood pressure can be suppressed by a mild action, and since this peptide is derived from soybean protein used as a food as described above, there are no problems such as side effects and the human body Was also very safe.

[Effect of the Invention] As described in detail above, the angiotensin-converting enzyme inhibitor according to the present invention can effectively inhibit the activity of angiotensin-converting enzyme that converts angiotensin I into angiotensin II with a mild action, and The isolated soybean protein obtained from soybean used as a food is derived from a food that can be produced by decomposing it with pepsin, and has no side effects and is highly safe.

As a result, the angiotensin converting enzyme inhibitor according to the present invention is highly safe for the human body, can be administered as a food or the like, can lower blood pressure with a mild action, and can also be expected to prevent hypertension. It was

[Brief description of drawings]

Each of the drawings is a diagram showing an embodiment of the present invention.
The figure shows the absorbance at 420 nm and the angiotensin-converting enzyme inhibition rate in each fraction fractionated by molecular sieve chromatography (Pharmacia Sephadex G-25). Fig. 2 shows the molecular sieve chromatography (Pharmacia). In each fraction fractionated by Sephadex G-10)
The figure showing the absorbance for 420 nm light and the angiotensin-converting enzyme inhibition rate, and FIGS. 3 (A) and (B) show the angiotensin-converting enzyme inhibition rate and the absorbance for 280 nm light in each fraction fractionated by cation exchange separation. Fig. 4 is a diagram showing the absorbance at 280 nm in the case of the first fractionation using high performance liquid chromatography, and Fig. 5 is the case of the second fractionation using high performance liquid chromatography. It is a figure which shows the light absorbency with respect to the light of 280 nm.

Continuation of front page (51) Int.Cl. ⁶ Identification number Office reference number FI Technical display area A61K 38/55 AED C07K 99:00

Claims (1)

[Claims] 1. An angiotensin-converting enzyme inhibitor containing a peptide represented by the following structural formula [1] or a salt thereof. [1] Asp-Gln-Thr-Pro-Arg-Val-Phe