JPS6151562B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to an angiotensin converting enzyme inhibitor having the following structure. Ala-Val-Pro-Tyr-Pro-Gln-Arg In vivo enzyme inhibitors previously found in actinomycete culture fluids have various effects such as anti-inflammatory, anti-peptic ulcer, and anti-cancer effects. It has been expected to be used as a medicine or research reagent. Regarding angiotensin converting enzyme inhibitors, several types of inhibitors produced by microorganisms have recently become known, including peptide inhibitors obtained from Brazilian snake venom and Japanese snake venom, and gelatin inhibitors. It has been known for some time that this product was isolated from a solution treated with collagenase derived from a microorganism. In addition, the Squibb Company of the United States has synthesized captopril, a proline derivative, which has a strong inhibitory effect and is attracting attention as a new orally available drug. However, since all of these inhibitors are expensive, it is desired to develop inhibitors derived from natural products that can be obtained at low cost and have fewer side effects. On the other hand, the present inventors had previously succeeded in obtaining an angiotensin converting enzyme inhibitor by decomposing cow-derived casein with trypsin etc.
58-109425, JP-A-59-44323, JP-A-59-
44324). This time, the present inventors succeeded in preparing a new angiotensin converting enzyme inhibitor having the above structure by treating bovine casein with trypsin etc. as in the previous case. The inhibitor of the present invention exhibits an inhibitory effect on angiotensin converting enzyme. In this case, angiotensin converting enzyme converts angiotensinogen, which is secreted by the liver, into angiotensin (Asp-Arg-Val
-Tyr-Ile-His-Pro-Phe-His-Leu), and this substance is converted into angiothecin (Asp).
-Arg-Val-Tyr-Ile-His-Pro-Phe). And this angiotensin ()
causes blood pressure to increase by contracting smooth muscle in blood vessel walls,
In addition to blood vessels, it also contracts the smooth muscles of the gastrointestinal tract and uterus, and also acts on the adrenal cortex to promote the secretion of aldosterone. Also,
Kallikrein, an enzyme present in plasma, breaks down a protein called kininogen to produce bradykinin, which dilates blood vessels and lowers blood pressure, but this bradykinin is broken down and inactivated by the action of angiotensin converting enzyme. Thus, angiotensin convertase generates a pressor peptide (angioncin) on the one hand, and degrades an antihypertensive peptide (bradykinin) on the other hand,
As a result, blood pressure increases. The inhibitor according to the present invention has an inhibitory effect on anditheonsine converting enzyme, which exhibits such an effect, and is particularly effective as an antihypertensive agent. In order to obtain the angiotensin converting enzyme inhibitor according to the present invention, bovine casein is decomposed with trypsin under conditions of pH 5.5 to 9.0, and the decomposed product is treated with trypsin at about 100°C or with the addition of an acid. and undegraded casein are precipitated, and this precipitate is removed by centrifugation or the like. After neutralizing the mother liquor thus obtained with an alkali such as sodium hydroxide, it is concentrated 2 to 3 times under reduced pressure. The concentrate thus obtained is purified to obtain a product. The present inhibitor can also be obtained using conventional peptide synthesis methods. That is, the amino group of one amino acid is protected with a benzyloxycarbonyl group or t-butoxycarbonyl group, the carboxyl group of the other amino acid or peptide is protected with a benzyl ester, etc.
Coupling with DCC (N,N'-dicyclohexylcarbodiimide) or the like. By repeating this operation, the protecting group is removed and the product can be purified and obtained. The inhibitor according to the present invention is in the form of a white powder at room temperature, and the R _f value of its aqueous solution determined by thin layer chromatography (silica gel plate, cellulose plate, ninhydrin coloring) is as shown in Table 1 below. Further, by dissolving it in 6M hydrochloric acid and performing hydrolysis at 110°C for 24 hours under vacuum, an amino acid mixture having the composition shown in Table 2 below can be obtained. The method of ingesting the angiotensin converting enzyme inhibitor of the present invention is generally performed by intravenous injection, for example,
An aqueous solution of the inhibitor is intravenously injected at a concentration of 0.01 to 1 mg of the inhibitor per 1 kg of animal. The angiotensin converting enzyme inhibitor according to the present invention can be applied to mammals that have the enzyme endogenously in their bodies, such as humans, rats, and dogs. Next, the present invention will be explained in more detail with reference to Examples. Example 2 g of bovine casein was suspended in 50 ml of 0.04 M phosphate buffer (PH7.4), 5 mg of trypsin (manufactured by PL Biochemicals, derived from pancreas) was added, and the mixture was incubated at 37°C.
Incubate overnight. After the reaction, the product has a final concentration of 0.5
Specified hydrochloric acid is added to denature and precipitate trypsin and undegraded casein. After removing the precipitate by centrifugation, neutralize the hydrochloric acid with sodium hydroxide to bring the pH to 7.0.
and concentrate the mother liquor 2-3 times under reduced pressure. Next, add the concentrate obtained above to
Purify by adding to LH20 column and eluting with distilled water. Then, the maximum active fraction at this time is collected and concentrated under reduced pressure. Note that the column processing conditions in this case are as follows. Column: Height 112 cm, inner diameter 3 cm Sample addition amount: 20 ml Flow rate: 1.2 ml/min Elution: Distilled water Next, the active fraction fractionated with the Cephadex LH-20 was added to the SP-Sephadex C.
-25 column and elute with a linear concentration gradient of 0 to 0.5M ammonium formate (PH = 7.0). The most active fraction is collected and concentrated under reduced pressure. In addition,
The column processing conditions in this case are as follows. Column: height 49cm, inner diameter 2cm Sample addition amount: 5ml Flow rate: 0.4ml/min Elution: 0-0.5M ammonium formate (PH=
7.0) Linear concentration gradient Next, the active fraction obtained above is added to a Sephadex LH-20 column and desalted. The column processing conditions in this case are as follows. Column: Height 64 cm, internal diameter 2 cm Flow rate: 0.4 ml/min Elution: Distilled water Next, the sample desalted using the Sephadex LH-20 described above is dried under reduced pressure to obtain a white powder substance (4 g of casein). (About 2mg obtained from). Next, the R ^f value of this substance by thin layer chromatography (silica gel plate and cellulose plate, ninhydrin coloring) was determined and was as follows.

[Table] In addition, the sample was dissolved in 6M hydrochloric acid and heated to 110% under vacuum.
After heating at ℃ for 24 hours, analysis was performed using an amino acid analyzer, and the following results were obtained.

[Table] Next, when the substance of the present invention was treated with carbocypetidase Y, Gln (glutamine) was detected. Therefore, it was detected by hydrochloric acid hydrolysis.
Glu (glutamic acid) is found in peptides.
Gln (glutamine). Next, the primary amino acid sequence of the substance of the present invention was determined using an amino acid analyzer using leucine aminopeptidase, carboxypeptidase A, and carboxypeptidase Y, and it was confirmed that the substance had the following structure. Ala-Val-Pro-Tyr-Pro-Gln-Arg Next, the following experiment was conducted to measure the enzyme inhibitory activity of the substance of the present invention. First, 5g of Rabbittran Langacetone powder was mixed with 50ml of 0.1M sodium borate buffer (PH=
8.3) and centrifuged at 40000 g for 40 minutes, and the supernatant was further diluted 5 times with the above buffer to obtain angiosin converting enzyme. Put 0.03ml of the sample containing the substance of the present invention into a test tube,
Add 0.25 ml of hipryl histidyl leucine (final concentration 5 mM, including 300 mM NaCl) as a substrate, and after incubating at 37°C for 10 minutes, add 0.1 ml of the above enzyme solution.
ml was added and reacted at 37°C for 30 minutes. after that,
After terminating the reaction by adding 0.25 ml of 1N hydrochloric acid, 1.5 ml of ethyl acetate was added, and the absorption value at 228 nm of hyperric acid extracted in ethyl acetate was measured, and this was taken as the enzyme activity. Note that under these conditions, the absorption value at 228 nm when the present inhibitor is not included is approximately 0.25. A plurality of such experiments were conducted, and the inhibition rate was calculated using the following formula. Inhibition rate = AB/A x 100 (%) A: Absorption value at 228 nm without inhibitor (0.25) B: Absorption value at 228 nm when inhibitor is added And when inhibition rate is 50% Inhibitor concentration I D ₅₀
As a result, the inhibitor of the present invention has a concentration of 1.5×10 ^-5 M
It was hot.

Claims (1)

[Claims] 1. An angiotensin converting enzyme inhibitor consisting of the following structure. Ala―Val―Pro―Tyr―Pro―Gln―Arg