JP2003306443A - Cholesterol-reducing peptide - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a cholesterol-reducing system using a new cholesterol- reducing peptide. <P>SOLUTION: This cholesterol-reducing agent contains peptides IIAE, IAEK, IIA, IAE, AEK and peptide EK as active ingredients. It is confirmed in in vivo tests that these peptides show excellent safety and high effects, and excellent food or drink and/or excellent medicine for reducing the cholesterol are provided. <P>COPYRIGHT: (C)2004,JPO

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to cholesterol reduction, and more particularly to a cholesterol reduction system using a novel cholesterol-reducing peptide. The peptide according to the present invention is used as a drug as a cholesterol-reducing agent, a cholesterol-reducing beverage, food, nutritional food,
It is used as a food or drink such as a functional food or a food for specified health use, and can be widely used for reducing cholesterol and preventing cholesterol accumulation.

[0002]

2. Description of the Related Art Recently, it has been pointed out that an increase in lifestyle-related diseases such as hypercholesterolemia has been pointed out, and various dietary fibers, soybean proteins, unsaturated fatty acids and other food ingredients have been studied to reduce cholesterol.

This point is no exception in the field of patents. For example, according to JP-A-60-11425, oligopeptides having a molecular weight of 200 to 1500 obtained by hydrolyzing soybean protein with an enzyme are used. It has been shown to have an effect of lowering intermediate cholesterol levels. As described above, plant proteins represented by soybean proteins and the like, and enzymatic degradation products thereof are generally considered to have a serum cholesterol lowering action and an anti-atherogenic action.

[0004]

SUMMARY OF THE INVENTION As described above, in view of the present state of the art in which research and development of serum cholesterol-lowering agents derived from soybean proteins are being actively conducted, the present inventors have
It was decided to develop a completely new novel cholesterol lowering agent that is highly effective and highly safe, and a completely new type of peptide cholesterol lowering agent that is not derived from soybean protein.

[0005]

Means for Solving the Problems As a result of investigations from various aspects in order to solve the above-mentioned problems, the present inventors synthesized many peptides focusing on peptides, particularly oligopeptides, and obtained the obtained peptides. Screened. As a result, 6 kinds of peptides (peptides IIAE, IAEK, IIA, IA) with excellent serum cholesterol concentration reducing action were obtained.
E, AEK and peptide EK) were discovered, and as a result of in vivo tests, it was confirmed that these peptides can be used as a cholesterol-reducing food or drink or medical agent, and the present invention was completed.

The peptides IIAE, IAEK, IIA, I according to the present invention
AE, AEK and peptide EK are the sequence numbers in the sequence listing.
Consisting of the amino acid sequences shown in 1, 2, 3, 4, 5 and 6,
Shown below. Peptide IIAE: (in case of single letter notation) IIAE; Ile-Ile-Al
a-Glu (in case of three-letter code) Peptide IAEK: (in case of single-letter code) IAEK; Ile-Ala-Gl
u-Lys (in case of three-letter code) Peptide IIA: (in case of single-letter code) IIA; Ile-Ile-Ala
(Three-letter notation) Peptide IAE: (One-letter notation) IAE; Ile-Ala-Glu
(Three-letter code) Peptide AEK: (One-letter code) AEK; Ala-Glu-Lys
(Three-letter notation) Peptide EK: (One-letter notation) EK; Glu-Lys (Three-letter notation)

That is, the present invention is characterized by the fact that a peptide having a specific amino acid sequence has an excellent cholesterol-reducing action, which was confirmed in an in vivo test. The amount used depends on symptoms, age, weight,
Although it varies depending on the administration method, dosage form, etc., normally, about 0.1 mg to 1,500 mg can be administered to an adult daily.

The active ingredient according to the present invention has no or very low toxicity and shows excellent safety, and 500 mg orally per day was orally administered to mice, but no acute toxicity was observed. Therefore, when using it as food and drink,
There is no particular limitation on the amount of the active ingredient used in any of preventive use, health use, and food / drink, and even when used as a medicine, it may be appropriately used within the above range depending on the patient. Further, since the present active ingredient does not exhibit remarkable acute toxicity even if it is taken in a large amount, it may be used in a larger amount than the above range if necessary.

Examples of the present invention will be described below.

[Example 1] The peptides of SEQ ID NOs: 1 to 6 were respectively synthesized using an automatic peptide synthesizer. The purity of the obtained peptides was confirmed to be 95% by reverse phase HPLC, respectively. These synthetic peptides were used to reduce serum cholesterol concentration in vivo in mice.
Confirmed by tests. Similarly, casein trypsin hydrolyzate (CTH) and trypsin hydrolyzate of β-lactoglobulin (LTH) were also subjected to in vivo tests. LTH was prepared as follows. First, 10 kg of β-lactoglobulin composition separated from sweet whey
It was dissolved in 0 kg of water and the liquid temperature was set to 42 ° C. Sodium hydroxide was added to this to adjust the pH to 7.5 to 8.0, and the pH was adjusted to 0.
Trypsin (NOVO 4500K) 100g dissolved in 01N HCl solution
Was added. Then, the mixture was enzymatically decomposed for about 3 hours while maintaining the pH, then immediately cooled to pH 6.9 to 7.0, and then deactivated by a plate sterilizer. By spray-drying this liquid, about 9.0 kg of β-lactoglobulin trypsin hydrolyzate (LTH) was obtained. A trypsin hydrolyzate of casein (CTH) was also prepared in the same manner.

ICR male mice having an initial body weight of about 20 g were fed with a commercially available solid feed for 3 days, and then 6 mice each were divided into groups. 20%
Casein trypsin hydrolyzate (CTH), β-lactoglobulin trypsin hydrolyzate (LTH), and peptides each containing 0.3% of peptide were added to the salmon containing 0.5% cholesterol and 0.25% sodium cholate added to casein. The added diet was allowed to freely ingest for 4 days. The diet composition is shown in Table 1.

[0012]

[Table 1]

In the above, the mineral is AIN-93G mineral mixture, and the above-mentioned feed contains the following minerals.
The following amount (mg) is contained per 1 kg.

(Mineral mixture) AIN-93G mineral mixture (mg / kg feed) Ca: 5000, P: 1561, K: 3600, S: 300, Na: 1019, Cl:
1571, Mg: 507, Fe: 35, Zn: 30, Mn: 10, Cu: 6, I:
0.2, Mo: 0.15, Se: 0.15, Si: 5, Cr: 1.0, F: 1.
0, Ni: 0.5, B: 0.5, Li: 0.1, V: 0.1.

(Vitamin mixture) AIN-93G vitamin mixture (mg / kg feed) all-trans-retinyl palmitate: 4000IU, cholecalcifer
ol: 1000IU, all-rac-α-tocopheryl acetate: 75IU, p
hylloquinone: 0.75, thiamine-HCl: 6.36, rivoflavi
n: 6.0, pyridoxin-HCl: 7.29, nicotinic acid: 30, C
a-pantothenate: 16.31, folic acid: 2.0, Cyanocobal
amin: 0.025, biotin: 0.2.

Mice were sacrificed by cardiac blood sampling. Serum
It was prepared by centrifugation at 3000 rpm for 15 minutes. Serum cholesterol was defined by an enzymatic method, specifically, using a commercially available kit (Cholesterol E-Test Wako; Wako Pure Chemical Industries, Ltd.). As the standard, (Presetset cholesterol; Boehringer Maiheim LLJ Nonouchi Co., Ltd.) was used. Similarly, HDL cholesterol was measured using HDL cholesterol test Wako (Wako Pure Chemical Industries, Ltd.). LDL + VLDL cholesterol was calculated. The obtained results are shown in Table 2 below.

[0017]

[Table 2]

In the above table, the feeding period was 4 days, each value was the average ± SEM of 6 animals in one group, and Duncan's multiple range test and Stude were used for statistical analysis of the experimental results.
nt's t-test was used. Note that * in the table is significantly different from the CTH result (* p <0.05, ** p <0.01, *** p
<0.001), indicating that there is a significant difference between different letters (p <0.05). In addition, LDL + VLDL cholesterol = total cholesterol-HDL cholesterol is shown.

As is clear from the above results, it was confirmed for the first time in the world that all of the six kinds of peptides significantly increase the arteriosclerosis index and exhibit serum cholesterol lowering action as compared with CTH. (Table 2).

[Example 2] Vitamin C 20 g, granulated sugar
To 50 g and 30 g of an equal mixture of corn starch and lactose, 50 g of peptide IIAE was added and mixed well. The mixture was divided into 100 equal parts and packed in a bag to produce 100 bags of 1.5 g of stick-shaped nutritional health food for cholesterol reduction.

[Example 3] Peptide IAEK (40 g) was used in place of peptide IIAE (50 g), and vitamin C (20 g) was used.
The same procedure as in Example 2 was repeated except that an equal amount mixture of vitamin C and citric acid (30 g) was used instead, and after sufficient drying, a nutritional health food for cholesterol reduction was produced.

Example 4 Peptide IIA powder 100 g, saccharide 150
g, honey 15g, ascorbic acid 1g, citric acid 0.5g, water to an appropriate amount of perfume to make 1kg, sterilize this at 95 ° C for 20 minutes, and aseptically fill each 100ml bottle, Made a health drink.

[Example 5] 20% aqueous solution of peptide IAE powder 2
00g, tocopherol acetate 5g, thiamine nitrate 10g, nicotinamide 20g, anhydrous caffeine 50g, benzoate and perfume.
Each was aseptically filled into pins to produce a health drink as a medicine.

Example 6 (1) Peptide AEK 50 g (2) Lactose 90 g (3) Corn starch 29 g (4) Magnesium stearate 1 g (1), (2) and (3) (however, 17 g) were mixed, (3)
(However, 7 g) was granulated together with the base prepared.
(3) (however, 5g) and (4) were added to the obtained condyle and mixed well, and this mixture was compressed with a compression tablet machine to give 1000 tablets containing 50 mg of active ingredient (1) per tablet. Was manufactured.

[Example 7] Peptide E instead of peptide AEK
Tablets were produced in the same manner as in Example 8 except that K was used. The dose varies depending on the patient's symptoms and age, but the dose is 0.1 to 1500 mg / kg / day once to four times a day. As described above, the peptide used in the present invention has almost no problem in safety, and therefore, it may be administered at a dose exceeding the above dose. When it is used as health maintenance, promotion, health care, nutritional supplement, etc., the dose smaller than the above dose may be taken for a long period of time.

[0026]

INDUSTRIAL APPLICABILITY According to the present invention, the serum cholesterol concentration can be reduced very efficiently and the safety is extremely high. Therefore, it is only useful as a preventive or therapeutic agent for reducing the serum cholesterol concentration. In addition, no unpleasant flavor such as strong bitterness is recognized, and it can be used as a preventive or therapeutic food or drink for cholesterol reduction.

[Sequence Listing] SEQUENCE LISTING <110> Meiji Dairies Corporation <120> Cholesterol Lowering Peptides <130> 02H012 <160> 6 <170> PatentIn Ver. 2.1 <210> 1 <211> 4 <212> PRT <213> Artificial Sequence <220><223> Description of Artificial Sequence: Cholester
ol Lowering Peptide <400> 1 Ile Ile Ala Glu 1 <210> 2 <211> 4 <212> PRT <213> Artificial Sequence <220><223> Description of Artificial Sequence: Cholester
ol Lowering Peptide <400> 2 Ile Ala Glu Lys 1 <210> 3 <211> 3 <212> PRT <213> Artificial Sequence <220><223> Description of Artificial Sequence: Cholester
ol Lowering Peptide <400> 3 Ile Ile Ala 1 <210> 4 <211> 3 <212> PRT <213> Artificial Sequence <220><223> Description of Artificial Sequence: Cholester
ol Lowering Peptide <400> 4 Ile Ala Glu 1 <210> 5 <211> 3 <212> PRT <213> Artificial Sequence <220><223> Description of Artificial Sequence: Cholester
ol Lowering Peptide <400> 5 Ala Glu Lys 1 <210> 6 <211> 2 <212> PRT <213> Artificial Sequence <220><223> Description of Artificial Sequence: Cholester
ol Lowering Peptide <400> 6 Glu Lys 1

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) C07K 5/083 C07K 5/10 5/10 A61K 37/02

Claims (6)

[Claims] 1. A cholesterol-reducing food or drink or medical agent, which comprises a peptide represented by the amino acid sequence of SEQ ID NO: 1 in the sequence listing as an active ingredient. 2. A cholesterol-reducing food or drink or medical agent, which comprises the peptide represented by the amino acid sequence of SEQ ID NO: 2 in the sequence listing as an active ingredient. 3. A cholesterol-reducing food or drink or medical agent, which comprises the peptide represented by the amino acid sequence of SEQ ID NO: 3 in the sequence listing as an active ingredient. 4. A cholesterol-reducing food or drink or medical agent, which comprises the peptide represented by the amino acid sequence of SEQ ID NO: 4 in the sequence listing as an active ingredient. 5. A cholesterol-reducing food or drink or medical agent, which comprises the peptide represented by the amino acid sequence of SEQ ID NO: 5 in the sequence listing as an active ingredient. 6. A cholesterol-reducing food or drink or medical agent comprising a peptide represented by the amino acid sequence of SEQ ID NO: 6 in the sequence listing as an active ingredient.