JP2003024012A - Angiotensin i converting enzyme inhibitor and antihypertensive functional food - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an angiotensin I converting enzyme inhibitor having as the active ingredient a peptide derived from a natural product and excellent in absorbency, to provide an antihypertensive functional food having as the active ingredient the peptide, and to provide a nutrient preparation having as the active ingredient the peptide. SOLUTION: The angiotensin I converting enzyme inhibitor has as the active ingredient a dipeptide such as Ala-Pro existing in a fermentation product obtained by fermenting a fish fresh hydrolyzate or in a fish and shellfish seasoning. The antihypertensive functional food having as the active ingredient the dipeptide and also the nutrient preparation having as the active ingredient the dipeptide are provided.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a dipeptide present in a fermentation product of a fish hydrolyzate, an angiotensin I converting enzyme inhibitor containing the dipeptide, a hypotensive functional food containing the dipeptide and the dipeptide. Concerning nutritional supplements.

[0002]

BACKGROUND OF THE INVENTION It is well known that the renin-angiotensin system is deeply involved in the development of hypertension. The renin-angiotensin system is an angiotensin I-converting enzyme present in vascular endothelial cell membranes and the like.
(EC3.4.15.1, hereinafter also referred to as ACE) plays an important role. In this case, ACE is angiotensin I (Asp-Arg-Val-) formed by cleavage of angiotensinogen secreted from the liver by the enzyme renin produced in the kidney.
Tyr-Ile-His-Pro-Phe-His-Leu), which acts as angiotensin II (Asp-Arg-Val-Tyr-Ile-His-Pro-Ph).
e). This angiotensin II has actions such as contraction of vascular smooth muscle to raise blood pressure, and further acts on adrenal cortex to promote secretion of aldosterone. On the other hand, the enzyme kallikrein existing in plasma cleaves the protein kininogen to produce bradykinin that dilates blood vessels and lowers blood pressure. This bradykinin is decomposed and inactivated by the action of ACE. Thus, ACE promotes blood pressure by degrading the pressor peptide angiotensin II on the one hand and degrading the blood pressure-lowering peptide bradykinin on the other hand. Therefore, it is possible to suppress an increase in blood pressure or decrease blood pressure by suppressing the activity of ACE. ACE inhibitors include several peptides obtained from snake venom, including captopril (D-3-merc
Many synthetic compounds such as apto-2-methylpropanoyl-L-proline) are known, and captopril and the like are used as antihypertensive drugs. Further, in recent years, a large number of ACE-inhibiting peptides have been found in various foods, of which milk casein, fermented milk, and foods to which peptides derived from bonito flakes are added have been put into practical use as foods for specified health use (Japanese Patent Publication No. 60-23085, Japanese Examined Sho 61-51562, Japanese Examined Sho 61-51564,
Biopolymers, 43, 119 (1997), Biopolymers, 43, 129
(1997)).

[0003]

As described above, a number of angiotensin I-converting enzyme inhibitors have been reported, but angiotensin I-converting enzyme which has higher absorbability into blood vessels and stability and which can be produced at a low cost. Inhibitors are always required not only as pharmaceuticals but also as materials for foods for specified health uses.

[0004]

The present inventors have found that the seafood seasoning has angiotensin I converting enzyme inhibitory activity and contains a large number of dipeptides (peptides having two amino acid residues). Peptides are generally hydrolyzed and unstable due to the action of a digestive enzyme (protease), and on the other hand, the larger the molecular weight, the less easily absorbed from the intestinal tract. However, it is known that the dipeptide has good absorbability, and the peptide having proline exhibits high stability because it shows resistance to the action of protease secreted in the digestive tract. Therefore, by purifying a dipeptide having such excellent properties and confirming its structure, a peptide having angiotensin I converting enzyme inhibitory activity and a dipeptide mixture having excellent absorbability were found, and the present invention was completed. It was

That is, the present invention is (1) Gly-Pro, Asn-Pr
o, Ala-Pro, Thr-Pro, Ala-Val, Gly-Val, Asp-Me
angiotensin I-converting enzyme inhibitor containing a dipeptide derived from one or more seafood selected from the group consisting of t and Asp-Leu, (2) angiotensin I-converting enzyme inhibitor containing Ala-Pro derived from seafood, (3 ) Gly-Pro, Asn-Pro,
(4) A hypotensive functional food containing a dipeptide derived from at least one seafood selected from the group consisting of Ala-Pro, Thr-Pro, Ala-Val, Gly-Val, Asp-Met and Asp-Leu, (4) Seafood (5) Gl, a blood pressure-lowering functional food containing Ala-Pro derived from
y-Pro, Asn-Pro, Ala-Pro, Thr-Pro, Ala-Val, Gl
y-Val, Asp-Met, Asp-Leu, Glu-Ala, Asp-Val, Gl
(6) Asn-Pro, Thr-Pro, Ala-Val, a nutritional supplement containing a dipeptide derived from one or more seafoods selected from the group consisting of u-Met, Phe-Glu, Glu-Ile, and Glu-Leu , Asp-Me
The present invention relates to a novel dipeptide having an amino acid sequence represented by t and Asp-Leu. Hereinafter, the present invention will be described in detail.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION The dipeptide of the present invention is produced by, for example, immersing fish and shellfish in water together with salt, and by autolysis, optionally adding a protease and promoting proteolysis to produce the dipeptide. The production of the dipeptide by this self-digestion is usually carried out at room temperature for 6 to 12 months without the addition of protease.
When a protease is added, the aging period can be shortened depending on the amount added. After the aging is completed, the mixture is filtered to obtain a dipeptide-containing supernatant. Examples of the raw material of seafood include sardines, mackerel, salmon, hokke, kichinago, mackerel, scallop, oyster, and clams, and sardines are particularly preferable. Alternatively, it can be isolated from the seafood seasoning thus produced. Further, the dipeptide of the present invention can also be obtained by enzymatically hydrolyzing and fermenting a protein of another organism containing the same amino acid sequence portion as the peptide in a manner similar to the above. The method for producing the dipeptide and the dipeptide mixture of the present invention from the seafood seasoning obtained from sardines as a raw material will be specifically described below.

The seafood seasoning is loaded on a gel filtration column and eluted with 30% methanol to collect a fraction having angiotensin I converting enzyme inhibitory activity. Next, this fraction is loaded on an ion exchange column, and after elution with distilled water, the active fraction eluted with a linear concentration gradient of 0 to 1 M sodium chloride is collected. The active fraction was subjected to high performance liquid chromatography (HPLC) loaded with a reverse phase column,
Fractionation is performed with a linear gradient of 0-32% acetonitrile containing 0.1% trifluoroacetic acid.

The collected active fraction and dipeptide-containing fraction are dried under reduced pressure to obtain the dipeptide and dipeptide mixture of the present invention having angiotensin I converting enzyme inhibitory activity.

The angiotensin I-converting enzyme inhibitory activity of the peptide of the present invention obtained as described above is described in "Studies on Biological Function Regulators in Foods (Biochemical Experimental Method
38) ”(published by the Academic Publishing Center). In other words, angiotensin I-converting enzyme with a buffer added to it is used as an enzyme solution or Hipp
A solution of uryl-His-Leu dissolved in a buffer was used as a substrate solution,
The sample solution of the present invention and the enzyme solution are mixed, the substrate solution is further added to cause an enzymatic reaction, and Hippuric acid (hippuric acid) released after the reaction is stopped is quantified by HPLC, and the inhibition rate is calculated. Good.

The dipeptide of the present invention can also be used as a salt. The dipeptide of the present invention or a salt thereof is used as it is, or is formulated into a tablet, a powder, a wettable powder, an emulsion, a capsule or the like with a commonly used solid carrier, liquid carrier, emulsifying dispersant or the like, and angiotensin I. It can be used as a converting enzyme inhibitor. As the carrier,
Water, gelatin, starch, magnesium stearate, lactose, vegetable oil and the like can be mentioned. Further, the dipeptide of the present invention or a salt thereof can be added to various foods to be used as functional foods, foods for specified health use and the like.
Examples of such foods include soft drinks, lactic acid drinks, seasonings, soups, cheeses, hams, confectionery and the like.

The above-mentioned dipeptide salts of the present invention are pharmaceutically acceptable acid addition salts and base addition salts.
As the pharmaceutically acceptable acid addition salt, for example, hydrochloric acid,
Sulfuric acid, nitric acid, salts with inorganic acids such as phosphoric acid; formic acid, acetic acid, propionic acid, glycolic acid, succinic acid, malic acid, tartaric acid, citric acid, salts with organic acids such as trifluoroacetic acid, and the like, In addition, pharmaceutically acceptable base addition salts include, for example, alkali metal salts such as sodium salts and potassium salts; alkaline earth metal salts such as calcium salts; amines such as ammonium, ethanolamine, triethylamine and dicyclohexylamine. Salt etc. are mentioned.

Examples of the use form of the angiotensin I converting enzyme inhibitor containing the dipeptide of the present invention as an active ingredient include parenteral administration such as intravenous injection and rectal administration, or oral administration.

The content of the dipeptide of the present invention in the angiotensin I converting enzyme inhibitor may be appropriately changed depending on its administration route and dosage form. For example, the content of the dipeptide of the present invention is 5 to 90% by weight based on the total weight of the preparation. %, Preferably 10
-60% by weight. When used by adding to a functional food or a food for specified health use, the dipeptide of the present invention or a mixture thereof is contained in the food in an amount of 0.01 to 50% by weight.

The dipeptide of the present invention is used as a dipeptide alone or a dipeptide mixture purified as described above, and also has a function of inhibiting angiotensin I-converting enzyme as a dipeptide-containing seafood seasoning without purification from the raw seafood seasoning. It can also be used as a food for specified health use, a functional food, or the like.

Further, the dipeptide mixture of the present invention can be used as a nutrient with good absorbability. The nutritional supplements include oral nutritional supplements and parenteral nutritional supplements. Parenteral nutritional supplements include infusions and enteral nutrition supplements. Hereinafter, the present invention will be described based on examples and test examples, but the present invention is not limited to these examples.

[0016]

Example 1 Purification of Angiotensin I-Converting Enzyme Inhibiting Dipeptide 10 ml of seafood seasoning was loaded on a Sephadex LH-20 column (Pharmacia, 26 × 900 mm) and eluted with 30% methanol to obtain angiotensin I. A fraction (290%) having a converting enzyme inhibitory activity (the measuring method is described in Test Example 1 below)
~ 300 ml fractions) were collected. Next, this fraction was loaded on an SP Toyopearl 650M column (Tosoh Corporation, 16 × 650 mm), and an active fraction (unadsorbed fraction) eluted with distilled water was collected. This was further loaded onto a DEAE Toyopearl 650M column (Tosoh Corporation, 16 x 650 mm) and eluted with a linear concentration gradient of active fraction A (unadsorbed fraction) and 0 to 1M sodium chloride eluted with distilled water. The active fraction B (position eluted with 0.08 M sodium chloride) was recovered.

The active fraction A was further analyzed by HPLC using a Symmetry C18 column (Waters 3.9 × 300 mm) to give 0.1
Fractionation was performed by linear gradient elution from 0-32% acetonitrile containing% trifluoroacetic acid. Active fractions (A-1, A-2, A-3, A, respectively) eluting at acetonitrile concentrations of 7%, 8%, 10%, 11%, 12%, 12.5%, and 15%.
-4, A-5, A-6, A-7) were obtained.

This active fraction B was further analyzed by HPLC using a Symmetry C18 column (Waters 3.9 × 300 mm) to give 0.1
Fractionation was performed by linear gradient elution from 0-32% acetonitrile containing% trifluoroacetic acid. Active fractions or dipeptide fractions (B-1, B-2, B-3, B, respectively) that elute around 4%, 12%, 14%, 15%, 18%, 19%, and 20% of acetonitrile concentration -4, B-5, B-6, B-7, B-8)
Got

The 15 kinds of dipeptides collected were dried under reduced pressure to obtain final products. The structures of these dipeptides are shown in Applied Biosystems Protein Sequencer 491.
When analyzed by type, the peptides A-1 to A-7 were Gly-Pro, Asn-Pro, Ala-Pro, Thr-Pro, and
Ala-Val, Gly-Val and Val-Pro. Also from B-1
B-8 dipeptides are Glu-Ala, Asp-Val, and
Asp-Met, Glu-Met, Phe-Glu, Glu-Ile, Asp-Leu,
It was Glu-Leu. Of the dipeptides B-1 to B-8, the only dipeptides showing angiotensin I converting enzyme inhibitory activity were Asp-Met and Asp-Leu.

Next, a chemically synthesized dipeptide having the same sequence as the above-mentioned dipeptide was subjected to the following conditions.
The retention time of the HPLC peak was compared with the retention time of the dipeptide obtained from the seafood seasoning. HPLC conditions: Column: Symmetry C18 (Waters 3.9 x 150 mm) Eluent: Linear gradient of 0 to 32% acetonitrile containing 0.1% trifluoroacetic acid (18 minutes) Flow rate: 1 ml / min Detection: 210 nm purple The retention time of each dipeptide chemically synthesized by external absorption was consistent with the retention time of each dipeptide purified from the seafood seasoning. Gly-Pro, Gly-Val, Ala-Pro, Val-Pr
The peptide represented by o is The Journal of Biological Ch
It is the same as the angiotensin I converting enzyme inhibitory peptide described in emistry, Vol.255, p.401 (1980).

Test Example 1 Measurement of angiotensin I-converting enzyme inhibitory activity The angiotensin I-converting enzyme inhibitory activity in Example 1 was measured by the following method. Rabbit lung-derived angiotensin I-converting enzyme purchased from Sigma was dissolved in a buffer solution (22 mM borate buffer solution, pH 8.3) to obtain an enzyme solution of 60 mU / ml. Also, Hip-His- purchased from Peptide Lab.
Leu (Hippuryl-L-histidyl-L-leucine) and sodium chloride were dissolved in the above borate buffer solution, respectively, 7.6 mM,
Concentration of 608mM (final concentration in the reaction mixture was 5mM and 40mM, respectively)
0 mM) and used as the substrate solution. In a 0.5 ml plastic tube, add 15 μl of sample solution and 50 of the above enzyme solution.
Add μl and incubate at 37 ℃ for 5 minutes.
5 μl was added and mixed well, and the reaction was carried out at 37 ° C. for 30 minutes. The reaction was then stopped by adding 20 μl of 10% trifluoroacetic acid. After the reaction was stopped, hippuric acid liberated by the enzymatic reaction was quantified by HPLC under the following conditions.

HPLC measurement condition column: μBondasphere 5 μC8 300Å (Waters Inc.)
3.9 x 150 mm) Eluent: linear gradient of 0-63% acetonitrile containing 0.1% trifluoroacetic acid (20 minutes) Flow rate: 1 ml / min Detection: UV absorption at 228 nm Repeated experiments like this, The inhibition rate was calculated by the following formula. Inhibition rate = (A−B) / A × 100 (%) [wherein, A: peak area of hippuric acid when the inhibitor is not included, B: peak area of hippuric acid when the inhibitor is added] The peptide concentration at which the above inhibition rate was 50% was expressed as an IC ₅₀ value. The IC ₅₀ values thus obtained are shown in Table 1.

Table 1 Peptide types IC ₅₀ value (μM) Gly-Pro 355 Asn-Pro 32 mM inhibition with 1.4 mM Ala-Pro 29 Thr-Pro 290 Ala-Val 15% inhibition with 790 μM Gly-Val 790 μM 17% inhibition Val-Pro 570 Asp-Met 600 Asp-Leu 37% inhibition at 790 μM

[0024]

INDUSTRIAL APPLICABILITY The present invention provides a dipeptide having angiotensin I converting enzyme inhibitory activity. The dipeptide of the present invention is derived from a natural product, can be easily obtained from seafood and seafood seasonings, and can be used as a raw material for medicines for treating or preventing hypertension and foods for specified health uses. Furthermore, the present invention can also be used as a nutritional supplement comprising a highly absorbable dipeptide.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61P 43/00 111 C07K 5/062 C07K 5/062 5/072 5/072 C12N 9/99 C12N 9/99 A61K 37/02 (72) Inventor Juon Niigata City, Niigata City, Tayuhama, Niigata 182, Shinwari, Mitsubishi Gas Chemical Co., Ltd. Niigata Research Center F-term (reference) 4B018 MD20 ME04 MF10 MF12 MF14 4C084 AA02 AA06 BA01 BA08 BA14 BA23 CA45 DC40 MA52 NA14 ZA422 ZC202 ZC212 4C087 AA02 BB29 MA52 NA14 ZA42 ZC20 ZC21 4H045 AA10 BA11 CA50 CA52 DA57 EA23 FA16 GA22 GA23 GA25

Claims (6)

[Claims] 1. Gly-Pro, Asn-Pro, Ala-Pro, Thr-Pr
An angiotensin I-converting enzyme inhibitor comprising at least one dipeptide derived from seafood selected from the group consisting of o, Ala-Val, Gly-Val, Asp-Met and Asp-Leu. 2. An angiotensin I converting enzyme inhibitor comprising Ala-Pro derived from seafood. 3. Gly-Pro, Asn-Pro, Ala-Pro, Thr-Pr
An antihypertensive functional food, which comprises at least one dipeptide derived from seafood selected from the group consisting of o, Ala-Val, Gly-Val, Asp-Met and Asp-Leu. 4. A hypotensive functional food comprising Ala-Pro derived from seafood. 5. Gly-Pro, Asn-Pro, Ala-Pro, Thr-Pr
o, Ala-Val, Gly-Val, Asp-Met, Asp-Leu, Glu-Al
a, Asp-Val, Glu-Met, Phe-Glu, Glu-Ile, Glu-Le
A nutritional supplement comprising at least one dipeptide derived from seafood selected from the group consisting of u. 6. Asn-Pro, Thr-Pro, Ala-Val, Asp-Me
A novel dipeptide having an amino acid sequence represented by t and Asp-Leu.