JP2003327543A - Vasodepressor activator and method of production for the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a vasodepressor activator and a method of production for the same manifesting an excellent vasodepressor activity, free from toxicity. <P>SOLUTION: The vasodepressor activator contains, as the active components, each peptide expressed by amino acid sequences described in sequence number 2, 12-15, 18-20 or 23 in the sequence table (refer to the specification), a mixture of peptides expressed by amino acid sequences described in sequence number 1-8 (refer to the specification) or a mixture of peptides expressed by amino acid sequences described in sequence number 9-23 (refer to the specification), or their pharmaceutically permissible salts and their salts permissible as foods. In the method of the present invention, the peptides, the active components, is produced by hydrolyzing animal milk casein using lactic acid bacteria- producing proteinase, and then purifying. <P>COPYRIGHT: (C)2004,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD OF THE INVENTION The present invention relates to an agent for lowering blood pressure.

[0002]

2. Description of the Related Art Conventionally, it has been known that fermented milk has various physiologically active actions, but it has not been well confirmed which component in fermented milk is involved in the activity (for example, Patent Document 1). ~ 3). On the other hand, casein trypsin, enzymatic degradation products such as pepsin, for example, blood pressure lowering activity,
It is also known that it exhibits physiological activity such as calcium solubilizing activity, but at present, little is known about the peptide contained in fermented milk and the function of the peptide. Lactic acid bacteria grow in milk, produce extracellular proteinase, and are considered to decompose milk proteins such as casein, and recently, some reports have been made on the cleavage site of casein by lactic acid bacterium-produced proteinase (for example, , Non-Patent Documents 1 to 2). However, there are still many unclear points regarding the cleavage site of casein, and no report has been made on the physiological function of the produced peptide. Further, in the case of peptides obtained by decomposing casein with trypsin, pepsin, etc., bitterness has been a serious problem.

[0003]

[Patent Document 1] JP-A-61-53216

[Patent Document 2] Japanese Patent Laid-Open No. 61-53217

[Patent Document 3] Japanese Patent Publication No. 3-64486

[Non-Patent Document 1] Monnet et al. (FEMS Microbiology Letter
s), 36 , 127-131 (1986)

[Non-Patent Document 2] Zevaco et al. (Le Lait), 68 , 393-408 (1988).

[0004]

SUMMARY OF THE INVENTION An object of the present invention is to provide a blood pressure lowering active agent which has no bitterness and toxicity and exhibits excellent blood pressure lowering activity.

[0005]

According to the present invention, each peptide represented by the amino acid sequence described in SEQ ID NO: 2, 12-15, 18-20, 23 of the sequence listing as an active ingredient, SEQ ID NO: 1- Containing the peptide mixture represented by the amino acid sequence described in 8 or the peptide mixture represented by the amino acid sequences described in SEQ ID NOs: 9 to 23, and a pharmaceutically acceptable salt and a food acceptable salt thereof. An antihypertensive agent is provided. Further, according to the present invention, animal milk casein is decomposed by a lactic acid bacterium-produced proteinase,
Each peptide purified, each peptide represented by the amino acid sequence described in SEQ ID NO: 2, 12-15, 18-20, 23, the peptide mixture represented by the amino acid sequence described in SEQ ID NO: 1-8, or SEQ ID NO: There is provided a method for producing the above-mentioned blood pressure lowering active agent, which comprises obtaining a peptide mixture represented by the amino acid sequences described in 9 to 23 and blending the obtained peptide or peptide mixture.

[0006] The present invention will be described in more detail below. Each peptide represented by the amino acid sequence described in SEQ ID NO: 2, 12 to 15, 18 to 20, 23 of the sequence listing used in the present invention, SEQ ID NO: 1
To produce a peptide mixture represented by the amino acid sequence described in ~ ~ or a peptide mixture represented by the amino acid sequence described in SEQ ID NOs: 9 to 23, for example, casein is decomposed by a lactic acid bacterium-produced proteinase, or a method for purification is usually used. It can be obtained by the chemical synthesis method or the like.

[0007] The lactic acid bacterium-produced proteinase is, for example, milk such as milk, goat milk, skim milk, or a medium for lactic acid bacteria, such as BL.
Medium, Briggs liver broth medium, MRS medium, GAM medium, TT
Y medium, MGLP medium, etc., are fermented with lactic acid bacteria, preferably cells are collected in the mid-logarithmic growth phase, and then washed with phosphate buffer or Tris-hydrochloric acid buffer containing calcium ions, etc. Extraction with non-phosphate buffer or Tris-HCl buffer or DEAE
-Proteinase and the like obtained by a method of purification using a Sepharose column, a gel filtration column or the like can be preferably mentioned.

[0008] The lactic acid bacterium when preparing the lactic acid bacterium-produced proteinase is preferably Lactococcus genus such as Lactococcus lactis JCM-5805, Lactobacillus helveticus JCM-1003.
(Lactobacillus helveticus JCM-1003), Lactobacillus casei subspecies casei JCM-1134 (Lactobaci
llus casei subsp.casei JCM-1134), Lactobacillus
Delphiki Subspecies Bulgaricus JCM-100
2 (Lactobacillus delbrueckii subsp.bulgaricus JCM-1
The genus Lactobacillus such as 002) and the genus Leuconostoc such as Leuconostoc lactis JCM-6123. Fermentation can be carried out preferably at 25 to 45 ° C for 3 to 12 hours. The resulting fermented milk usually has a pH of 3 to 4,
In order to increase the yield of the target proteinase,
It is preferable to carry out the fermentation while keeping the pH in a neutral range. Further, the extraction can be performed by repeating the extraction step preferably at 5 to 40 ° C. for 10 to 60 minutes 2 to 5 times.

[0009] To decompose casein with the lactic acid bacterium-produced proteinase, the lactic acid bacterium-produced proteinase is mixed with casein dissolved in a buffer such as a phosphate buffer,
The mixture is reacted at ~ 45 ° C for 1 to 12 hours, then centrifuged, preferably ultrafiltered with an ultrafiltration membrane having a molecular weight fraction of 10,000 to 50,000, and further purified by reverse phase liquid column chromatography. Depending on the method etc., SEQ ID NO: 1 to 23
(The peptides represented by the amino acid sequences described in Peptides (1) to (23) can be purified. In this case, the mixing ratio of lactic acid bacterium-produced proteinase and casein is 1:10 to 1000 by weight. Is preferred.

The blood pressure lowering active agent of the present invention comprises the peptide (1)
~ (23) peptide (2), (12) ~ (15), (18) ~ (2
0), (23), peptide (1) to (8) mixture or peptide (9) to
(23) A mixture, a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable salt thereof as active ingredients, which exhibit at least blood pressure lowering activity.

[0011] In the blood pressure lowering active agent of the present invention, the content ratio of the active ingredient is 0.1 to 100% by weight, particularly 0.5 to 10% by weight.
Is preferred.

The dosage form of the blood pressure lowering active agent of the present invention can be mainly administered orally. The dosage forms are tablets, granules,
It can also be used as a capsule or the like, or as a liquid preparation. Further, the active ingredient can be used by adding and blending it with ordinary medicines or medical foods, and also general foods. The dose of the blood pressure lowering active agent of the present invention varies depending on the patient's age, symptoms, etc., but is based on the above-mentioned active ingredient.
It is preferable to use 1 mg / kg body weight / day or more.

[0013] The blood pressure lowering active agent of the present invention may be blended with an excipient such as lactose and dextrin, a stabilizer and the like in addition to the above-mentioned active ingredients.

[0014]

The blood pressure lowering agent of the present invention is not toxic and exhibits excellent blood pressure lowering activity.

[0015]

EXAMPLES The present invention will now be specifically described based on examples, but the present invention is not limited to these. Example 1 Lactobacillus helveticus JCM-1003 was cultivated while maintaining pH at 6.0 in 9 wt% skim milk, and turbidity (absorbance at 590 nm)
At 1.0, 1% by weight of sodium citrate was added and the mixture was kept at room temperature for 20 minutes. Next, the cells were collected by centrifugation at 5000 rpm for 20 minutes, washed with 20 mM calcium chloride, 50 mM β-sodium glycerophosphate buffer (pH 8.0), and then added with 50 ml of 50 mM Tris-hydrochloric acid (pH 8.0). Extraction was carried out at 37 ° C for 30 minutes. Then, centrifugation was performed at 10,000 rpm for 10 minutes to collect a supernatant. The same operation was performed 4 times in total, and about 200 ml of the supernatant was collected (crude extract). This crude extract was passed through a DEAE-Sepharose column (5 ml) equilibrated with a 5 mM ethylenediaminetetraacetic acid solution (EDTA) and 20 mM Tris-hydrochloric acid buffer (pH 7.8, TE buffer) in advance. Wash the column with 30 ml of TE buffer containing 0.3 M sodium chloride, then add 1.0 M sodium chloride
Elute with 15 ml of TE buffer, and extract from this active fraction (eluting fraction)
150 μg of lactic acid bacterium-produced proteinase was obtained as almost a single protein.

[0016] Next, 1 g of casein dissolved in a 20 mM phosphate buffer (pH 7.5) was mixed with 50 μg of trypsin (manufactured by Wako Pure Chemical Industries, Ltd.) obtained above as a proteinase or as a comparison, and at 40 ° C for 5 hours. It was made to react. 10000 each reaction
After rotation and centrifugation for 10 minutes, ultrafiltration (trade name "Advantech Toyo UHP-150", ultrafiltration membrane: molecular weight fraction 10000, manufactured by Fuji Filter Industry Co., Ltd.) was performed, and proteinase-degrading peptide from 1 g of casein was obtained. About 700 mg (Yield about 70
%) And about 800 mg of tryptic peptides (yield about 80%) were obtained in the filtrate.

Next, using the casein-degrading peptide mixture thus obtained, the blood pressure lowering effect on spontaneously hypertensive rats (SHR rats, Charles River Japan) was examined. Each of the casein-degrading peptides was forcibly administered (15 mg / kg each) to 15-week-old male rats (5 rats per group) by gastric sonde, and the time course of blood pressure was compared with that of the non-administered group. For blood pressure measurement, a non-invasive blood pressure measurement device (trade name "PE-300", manufactured by Narco Biosystems) was used to determine the maximum blood pressure by the tail-cuff method. The results are shown in Figure 1.

From the results of FIG. 1, it was confirmed that the peptide containing the active ingredient of the present invention shows a significant blood pressure lowering effect after about 4 to 7 hours after oral administration. Such a strong effect was not observed with the tryptic peptide.

Example 2 The proteinase-decomposed casein degradation product obtained in Example 1 was further purified by high performance liquid chromatography (HPLC). The purification was performed using a column packed with reverse phase resin (M
& S PACK C-18, 0.46ψ × 150mm), pass the casein degradation product, wash with 0.1 wt% TFA aqueous solution, and then wash with 0.1 wt% TFA aqueous solution to 0.06 wt% TFA / (acetonitrile: isopropanol = 3: 7) solution. Elution was performed with a linear concentration gradient up to%.
The flow rate was 1 ml / min and the concentration gradient was 1% / min. The main absorption peak at 215 nm was collected respectively. Furthermore, the solvent was removed from each of these peptides, and further purification was performed by rechromatography under the same conditions. For each peptide,
Acetonitrile was removed under reduced pressure, and the peptide was obtained by freeze-drying. For these peptides, 120 with 6N hydrochloric acid
After hydrolysis at 24 ° C. for 24 hours, amino acid analysis (high-speed amino acid analyzer, trade name “MLC-203 type”, manufactured by Ato Co., Ltd.) was performed. The positions within α-casein and β-casein were identified by amino acid analysis. These peptides are α-1 to α-casein shown in Table 1 in the order of elution by HPLC, respectively.
8 and β-1 to 15 peptides were confirmed. Antihypertensive activity (ACEI activity), calcium solubilizing activity (CS activity) and antioxidant activity (SOD-like activity) of these peptides and casein hydrolysates degraded by proteinase prepared in Example 1 (peptide mixture), It measured according to the method shown in. For comparison, the activities of α-casein and β-casein trypsin degradation products were also examined in the same manner. The results are shown in Table 2.

<Angiotensin converting enzyme inhibition (ACEI) activity> (Method for measuring ACEI activity) 20 μl of a sample containing a peptide and 5 mM
Hiproil-His-Leu (HHL, Sigma), 260 μl of 0.3 M NaCl, 0.1 M borate buffer (pH 8.3) is kept in a test tube at 37 ° C. for 10 minutes. Then 0.05 U / ml angiotensin converting enzyme
(ACE: Sigma) (20 μl) was added, and the mixture was reacted at 37 ° C. for 30 minutes. Then, 250 μl of 1N hydrochloric acid was added to stop the reaction.
Add 1.7 ml of ethyl acetate and stir for 20 seconds.
Centrifugation was performed for 0 minutes to collect 1.4 ml of an ethyl acetate layer. After heating the ethyl acetate at 120 ℃ for 30 minutes and drying, add 1 ml of distilled water and stir for 20 seconds to absorb the extracted HHL (absorbance at 228 nm).
Was measured.

[0021] The inhibition rate was calculated by the following formula.

[Equation 1] A: Absorbance at 228 nm without sample B: Absorbance at 228 nm with sample added C: Absorbance at 228 nm without added enzyme and sample The sample required to inhibit the enzyme activity of ACEI by 50% concentration
(μg / ml) is given as IC ₅₀ .

<Calcium Solubilization (CS) Activity> (Method for Measuring CS Activity) Quantification of calcium is performed by a chelate method.
(Orthocresolphthalein complexone, OCPC method)
Went by. 50 μl of the sample containing the peptide is mixed with 10 μl of 20 mM calcium chloride and 10 mM Tris-hydrochloric acid buffer (pH 7.0), and the mixture is kept at room temperature for 5 minutes. Further, 40 μl of 20 mM phosphate buffer (pH 7.0) is added, and the mixture is kept at 37 ° C. for another 30 minutes.
After that, centrifugation was performed at 15,000 rpm for 5 minutes to collect 10 μl of the supernatant, and 800 μl of the buffer solution contained in the trade name `` Calcium C-Test Wako '' (Wako Pure Chemical Industries) and 80 μl of the enclosed OCPC reagent were added to develop color, The absorbance at 570 nm was measured.

[0023]

[Equation 2] A: Absorbance at 570 nm without addition of phosphate buffer B: Absorbance at 570 nm with sample added C: Absorbance at 570 nm without sample added Necessary for 50% solubilization of calcium The concentration of the sample (μg / ml) is shown in Table 2 as SC ₅₀ .

<Superoxide dismutase (SOD) -like activity> (Measurement of SOD-like activity) Diethylenetriamine pentaacetic acid (DE
TAPAC) solution (5 mg DETAPAC, 9.8 ml 50 mM potassium phosphate buffer, 0.37 ml 4 μg / ml catalase, 0.37 ml 1.83 mg
/ Ml nitro blue tetrazolium (NBT), 1.26 ml 1.0 mM
Xanthine) (400 μl), a peptide-containing sample (20 μl), and xanthine oxidase (manufactured by Sigma, 400-fold diluted) (50 μl) were mixed and incubated at 30 ° C. At this time, the difference in absorbance (absorbance at 560 nm) that changed over 3 minutes was measured. The inhibition rate of xanthine oxidase was calculated by the following formula.

[0025]

[Equation 3] A: Absorbance at 560 nm when sample is not added B: Absorbance at 560 nm when sample is added C: Absorbance at 560 nm when enzyme is not added Concentration of sample required to inhibit the enzyme activity of xanthine oxidase by 50% ( μg / ml) is shown in Table 2 as IC ₅₀ .

[0026]

[Table 1]

[0027]

[Table 2]

[Sequence list]                           SEQUENCE LISTING <110> Calpis Co., Ltd. <120> Agent having hypotensive activity and method for preparing the sam e <130> P03-352 <160> 23 <210> 1 <211> 4 <212> PRT <213> Bos taurus <400> 1 Ala Tyr Pro Ser 1 <210> 2 <211> 6 <212> PRT <213> Bos taurus <400> 2 Ala Tyr Phe Tyr Pro Glu 1 5 <210> 3 <211> 8 <212> PRT <213> Bos taurus <400> 3 Val Ala Pro Phe Pro Gln Val Phe 1 5 <210> 4 <211> 8 <212> PRT <213> Bos taurus <400> 4 Gly Ala Trp Tyr Tyr Val Pro Leu 1 5 <210> 5 <211> 14 <212> PRT <213> Bos taurus <400> 5 Gln Leu Asp Ala Tyr Pro Ser Gly Ala Trp Tyr Tyr Val Pro 1 5 10 <210> 6 <211> 30 <212> PRT <213> Bos taurus <400> 6 Gly Thr Gln Tyr Thr Asp Ala Pro Ser Phe Ser Asp Ile Pro Asn Pro 1 5 10 15 Ile Gly Ser Glu Asn Ser Glu Lys Thr Thr Met Pro Leu Trp             20 25 30 <210> 7 <211> 7 <212> PRT <213> Bos taurus <400> 7 Gly Ser Glu Asn Ser Glu Lys 1 5 <210> 8 <211> 4 <212> PRT <213> Bos taurus <400> 8 Gly Ser Glu Asn 1 <210> 9 <211> 7 <212> PRT <213> Bos taurus <400> 9 Lys Ala Val Pro Tyr Pro Gln 1 5 <210> 10 <211> 6 <212> PRT <213> Bos taurus <400> 10 Ala Val Pro Tyr Pro Gln 1 5 <210> 11 <211> 5 <212> PRT <213> Bos taurus <400> 11 Gln Ser Leu Thr Leu 1 5 <210> 12 <211> 15 <212> PRT <213> Bos taurus <400> 12 Lys Tyr Pro Val Gln Pro Phe Thr Glu Ser Gln Ser Leu Thr Leu 1 5 10 15 <210> 13 <211> 8 <212> PRT <213> Bos taurus <400> 13 Ser Lys Val Leu Pro Val Pro Glu 1 5 <210> 14 <211> 18 <212> PRT <213> Bos taurus <400> 14 Pro Pro Gln Ser Val Leu Ser Leu Ser Gln Ser Lys Val Leu Pro Val Pro Glu 1 5 10 15 <210> 15 <211> 8 <212> PRT <213> Bos taurus <400> 15 Gly Pro Val Arg Gly Pro Phe Pro 1 5 <210> 16 <211> 28 <212> PRT <213> Bos taurus <400> 16 Leu Pro Gln Asn Ile Pro Pro Leu Thr Gln Thr Pro Val Val Val Pro 1 5 10 15 Pro Phe Leu Gln Pro Glu Val Met Gly Val Ser Lys             20 25 <210> 17 <211> 54 <212> PRT <213> Bos taurus <400> 17 Leu Ser Ser Ser Glu Glu Ser Ile Thr Arg Ile Asn Lys Lys Ile Glu 1 5 10 15 Lys Phe Gln Ser Glu Glu Gln Gln Gln Thr Glu Asp Glu Leu Gln Asp             20 25 30 Lys Ile His Pro Phe Ala Gln Thr Gln Ser Leu Val Tyr Pro Phe Pro         35 40 45 Gly Pro Ile Pro Asn Ser     50 <210> 18 <211> 82 <212> PRT <213> Bos taurus <400> 18 Leu Ser Ser Ser Glu Glu Ser Ile Thr Arg Ile Asn Lys Lys Ile Glu 1 5 10 15 Lys Phe Gln Ser Glu Glu Gln Gln Gln Thr Glu Asp Glu Leu Gln Asp             20 25 30 Lys Ile His Pro Phe Ala Gln Thr Gln Ser Leu Val Tyr Pro Phe Pro         35 40 45 Gly Pro Ile Pro Asn Ser Leu Pro Gln Asn Ile Pro Pro Leu Thr Gln     50 55 60 Thr Pro Val Val Val Pro Pro Phe Leu Gln Pro Glu Val Met Gly Val 65 70 75 80 Ser Lys <210> 19 <211> 27 <212> PRT <213> Bos taurus <400> 19 Asp Glu Leu Gln Asp Lys Ile His Pro Phe Ala Gln Thr Gln Ser Leu 1 5 10 15 Val Tyr Pro Phe Pro Gly Pro Ile Pro Asn Ser             20 25 <210> 20 <211> 8 <212> PRT <213> Bos taurus <400> 20 Arg Asp Met Pro Ile Gln Ala Phe 1 5 <210> 21 <211> 14 <212> PRT <213> Bos taurus <400> 21 His Lys Glu Met Pro Phe Pro Lys Tyr Pro Val Gln Pro Phe 1 5 10 <210> 22 <211> 17 <212> PRT <213> Bos taurus <400> 22 Tyr Gln Gln Pro Val Leu Gly Pro Val Arg Gly Pro Phe Pro Ile Ile Val 1 5 10 15 <210> 23 <211> 19 <212> PRT <213> Bos taurus <400> 23 Leu Leu Tyr Gln Gln Pro Val Leu Gly Pro Val Arg Gly Pro Phe Pro 1 5 10 15 Ile Ile Val

[Brief description of drawings]

FIG. 1 is a graph showing the relationship between systolic blood pressure measured by the tail-cuff method and the time after administration in Example 1.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) // C07K 5/103 ZNA C07K 7/06 7/06 7/08 7/08 14/47 14/47 C12P 21/06 C12P 21/06 A61K 37/16 F term (reference) 4B064 AG01 CA21 CB06 CD20 CD25 CE03 CE06 CE10 DA01 DA10 4C084 AA02 BA01 BA08 BA16 BA17 BA18 BA19 BA20 CA04 CA59 DC23 DC32 DC40 MA52 NA14 ZA42 ZC20 4H045 AA10 AA20 BA09 BA14 BA15 BA16 BA17 BA18 BA21 CA43 DA57 EA01 EA23 FA70 GA10 GA23

Claims (3)

[Claims] 1. SEQ ID NOS: 2 and 12 in the sequence listing as active ingredients
~ 15, 18-20, each peptide represented by the amino acid sequence described in 23, the peptide mixture represented by the amino acid sequence described in SEQ ID NO: 1-8 or the amino acid sequence described in SEQ ID NO: 9-23 A blood pressure lowering active agent, which comprises a peptide mixture, and a pharmaceutically acceptable salt thereof and a food acceptable salt thereof. 2. Animal milk casein is decomposed and purified by a proteinase produced by lactic acid bacterium, and SEQ ID NO: 2, 12 to 15, 18 to 2 in the sequence listing.
0,23 to obtain each peptide represented by the amino acid sequence, the peptide mixture represented by the amino acid sequence represented by SEQ ID NO: 1-8 or the peptide mixture represented by the amino acid sequence represented by SEQ ID NO: 9-23 2. The method for producing an antihypertensive agent according to claim 1, wherein the obtained peptide or peptide mixture is blended. 3. The lactic acid bacterium-produced proteinase is milk or lactic acid bacterium growth medium, Lactococcus lactis,
Fermented with a lactic acid bacterium selected from the group consisting of Lactobacillus helveticus, Lactobacillus casei subspecies casei, Lactobacillus delbrukii subspecies bulgaricus, leuconostoc lactes and mixtures thereof. 3. The method according to claim 2, wherein the proteinase is produced by the lactic acid bacterium.