KR100318553B1 - New Bioactive Peptides with Angiotensin-1-Converting Enzyme Inhibitory Activity - Google Patents

Abstract

The present invention relates to a novel bioactive peptide Leu-Gln-Trp derived from human alphas1 casein having an inhibitory activity of angiotensin-I-converting enzyme (ACE) and its use for the treatment of hypertension. Synthesis of Leu-Gln-Trp, the 168-170th peptide of alpha s1 casein, followed by reverse phase-high performance liquid chromatography (RP-HPLC) to analyze and quantify the ACE inhibitory activity of the peptide As a result, the peptide has an ACE inhibitory activity and has an excellent effect useful as a therapeutic agent for hypertension.

Description

Novel Bioactive Peptides with Inhibitory Activity of Angiotensin-I-Converting Enzymes

The present invention relates to novel bioactive peptides having angiotensin-I-converting enzyme (ACE) inhibitory activity. More specifically, the present invention relates to novel bioactive peptides derived from human alphas1 casein having angiotensin-I-convertase inhibitory activity.

ACE is an enzyme that produces angiotensin-II, which acts as a potent vasoconstrictor, and induces blood pressure elevation by breaking down the vasodilator, bradykinin. Therefore, research has been continued to develop ACE inhibitors for the treatment of hypertension. Captopril, the most widely used oral medication for hypertension, has strong ACE inhibitory effects but has side effects that cause nail disease (Brueggermeyer, et. Al., Lancet, 1, 1352, 1984). In order to overcome this problem, efforts have been made to find peptides having ACE inhibitory activity in natural products, especially foods, and various biologically active peptides are derived from hydrolysates of food proteins (milk, milk, soybeans, wheat and tuna fish). (Yamamoto, N., Biopolymers, 43, 129, 1997). Biologically active peptides derived from food proteins are safe for the human body even if their activity is somewhat low, so it is expected that the side effects of continuous drug use can be eliminated.

Problems that arise when using peptides with ACE inhibitory activity as oral administration agents are loss of activity due to degradation of protease, disorders of absorption in the intestine. However, animal experiments have shown that a significant number of peptides pass through the barrier of digestion or intestinal absorption in the digestive tract and conclude that the absorption efficiency of di- or tri-peptides is superior to free amino acids. Accordingly, the present inventors have found a new shorter and more active peptide, which is a tri-peptide consisting of three amino acids of Leu-Gln-Trp and can be easily absorbed in the intestine without undergoing degradation of the protease.

An object of the present invention is to provide a novel bioactive peptide Leu-Gln-Trp derived from human alpha s1 casein having angiotensin-I-convertase inhibitory activity. Another object of the present invention is to provide a novel peptide Leu-Gln-Trp having angiotensin-I-convertase inhibitory activity for the treatment of hypertension.

The object of the present invention was achieved by synthesizing peptides 168-170 of human alpha s1 casein and analyzing and quantifying ACE inhibitory activity using reverse phase high performance liquid chromatography.

Hereinafter, the configuration and operation of the present invention will be described in detail.

1A, 1B and 1C show reverse phase-high performance liquid chromatography of angiotensin-I-converting enzyme (ACE) inhibitory activity of the Leu-Gln-Trp peptide. Analyzed.

Figures 2a and 2b is a graph showing the quantitative angiotensin-I-convertase inhibitory activity of the Leu-Gln-Trp peptide.

The present invention comprises adding a hydrochloric acid solution to the Hip-His-Leu substrate solution, and then adding ACE to inactivate the ACE enzyme activity, followed by enzymatic reaction with the substrate; Comprising the enzyme reaction compared to the enzyme reaction by adding ACE to the Hip-His-Leu substrate solution to terminate the reaction by adding sodium chloride solution; Unlike the enzyme reaction, peptide-Leu-Gln-Trp is added to Hip-His-Leu substrate solution, ACE is added to enzymatic reaction, and then sodium chloride solution is added to terminate the reaction; Analyze and quantify the amount of hyplic acid produced by the enzymatic reaction of the ACE step by reverse phase high performance liquid chromatography.

Hereinafter, the specific method of the present invention will be described step by step with reference to Examples, but the scope of the present invention is not limited only to these Examples.

Example 1 Investigation of ACE Inhibition Activity Using Substrate Solution Containing Hip-His-Leu

ACE inhibition was measured by using hippuryl-L-histidyl-L-leucine (Hip-His-Leu) as a substrate (Cushman, D. W. & Cheung, H. S., Biochem. Phamacol. 20, 1637, 1971).

Step 1: Enzyme Reaction Inactivating ACE

A 5 mM Hip-His-Leu (Sigma) substrate was dissolved in 0.1 M boric acid buffer solution (borate buffer pH8.3, 0.3 M sodium chloride) to adjust the total volume to 95 μL. 100 μL of 1 N hydrochloric acid solution was added to the solution, and 5 μL of 5 mU ACE (Sigma) solution was added thereto and reacted at 37 ° C. in a constant temperature bath for 30 minutes. Herein, 1N hydrochloric acid solution was performed to completely inactivate ACE before the reaction to completely block the conversion of the substrate.

Stage 2: Maximum Enzyme Activity of ACE

5 μL of 5 mU ACE solution was added to the substrate solution used in the first step, reacted in a constant temperature water bath at 37 ° C. for 30 minutes, and 1 N sodium chloride solution was added to terminate the reaction. In this step, since ACE is sufficient to convert the substrate to the product, the ACE activity of the reaction was set to 100%, and the ACE inhibitory ability of the peptide Leu-Gln-Trp performed in step 3 below was measured.

Step 3: ACE Enzyme Reaction Using Substrate Solution Added Peptide Leu-Gln-Trp

In this step, a chemically synthesized peptide Leu-Gln-Trp (Genemed) was added to the substrate solution mentioned in step 1 at various concentrations, and the same procedure as in step 2 was repeated.

Step 4: Quantitative Analysis of ACE Inhibitory Activity of Peptide Leu-Gln-Trp

The ACE activity of the first, second and third stages was determined by analyzing the amount of the produced hippuric acid by reverse phase high performance liquid chromatography (RP-HPLC). RP-HPLC analysis conditions were performed at 228 nm using a C18 (YMC, 4 μm, 4.6 x 250 mm) column with 0-60% acetonitrile containing 0.1% fluoroacetic acid at a rate of 1 mL per minute for 20 minutes. Absorbance was measured. As a result, Figure 1a is a result of analyzing the amount of the hyplic acid produced by inactivating the ACE in the first step and then reacted with the substrate, Figure 1b is a reaction of the produced hyplic acid after sufficiently reacting the ACE solution and the substrate solution Figure 1c is the result of the analysis of the amount of hypurilic acid produced by the addition of Leu-Gln-Trp in various concentrations and the enzyme reaction to the substrate solution and the ACE inhibitory ability of the peptide Leu-Gln-Trp is Calculated based on

Inhibition Rate (%) = × 100

Eb: amount of hyplic acid produced by the first stage enzymatic reaction.

Ec: the amount of hyplic acid produced by the second stage enzymatic reaction.

Es: amount of hyplic acid produced by enzymatic reaction with the addition of the third stage peptide Leu-Gln-Trp.

The results according to the above calculations are shown in FIGS. 2A and 2B, and the concentration of peptide Leu-Gln-Trp, ie, IC ₅₀ value, was ₅₀ when the inhibition rate was 50%.

The present invention provides Leu-Gln-Trp Nos. 168-170 as a novel physiologically active peptide derived from human alpha s1 casein having ACE inhibitory activity as described above, and Leu-Gln-Trp is a potent ACE (IC ₅₀ = 3.8 μM) has an inhibitory activity and thus has a therapeutic effect on hypertension, which is a very useful invention in the pharmaceutical industry.

Claims (1)

A composition for treating hypertension, comprising a bioactive peptide of the amino acid sequence of Leu-Gln-Trp derived from human alpha sl casein and having an angiotensin-I-converting enzyme (ACE) inhibitory activity.