JP3441411B2 - Cholesterol-lowering peptide - Google Patents

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 cholesterol-reducing agent as a drug, and also as a food or drink such as a cholesterol-reducing beverage, food, nutritional food, functional food, food for specified health use, for reducing cholesterol. In addition, it can be widely used for prevention of cholesterol accumulation.

[0003]

BACKGROUND OF THE INVENTION Sterols and their derivatives are essential constituents of the living body. Although triacylglycerol only plays a role as an energy storage, cholesterol and its fatty acid ester are important constituents of cell membranes, and at the same time, various important substances such as bile acid, steroid hormones and vitamin D in the body. This gives rise to a functional derivative. Cholesterol metabolism control is important for keeping the cardiovascular system healthy, and its abnormality causes arteriosclerosis.

In recent years, dietary fiber, soybean protein,
The positive effects of various food ingredients such as unsaturated fatty acids on cholesterol metabolism have been actively studied in Japan and abroad.

[0005] For example, Carroll and Hamilton have studied dietary proteins with respect to plasma cholesterol levels, and have revealed that soybean proteins lower plasma cholesterol levels compared to casein proteins (KK Carrol a.
nd RMG Hamilton, J. Food Sci., 40, 18-23 (197
Five)).

Further, according to JP-A-60-11425,
It has been shown that oligopeptides having a molecular weight of 200 to 1500 obtained by hydrolyzing soybean protein with an enzyme have a blood cholesterol concentration lowering action. 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.

[0007]

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
The development of a completely new, highly effective and highly safe cholesterol lowering agent, and the development of a completely new type of peptide cholesterol lowering agent which is not derived from soybean protein, has been set as a new technical subject.

[0008]

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, two types of peptides (peptide L and peptide I) having an excellent action of reducing serum cholesterol concentration were discovered, and as a result of further investigation, it was confirmed that they can be used as cholesterol-lowering agents or reduced foods and beverages, and the present invention was completed. .

Peptides L and I according to the present invention consist of the amino acid sequences shown in SEQ ID NOS: 1 and 2 of the Sequence Listing, respectively, and are shown below. Peptide L: Leu-Asp-Ile-Gln-Lys Peptide I: Ile-Ile-Ala-Glu-Lys These peptides have never been synthesized or isolated, and much less known to have a cholesterol-reducing action. Although not available, it can be easily synthesized and obtained by an automatic synthesizer or the like.

As will be apparent from the examples described below, these peptides have an excellent cholesterol-reducing action. Therefore, foods and drinks, foods and drinks for specified health uses, health drinks, health foods, and nutritional foods that utilize this action. In addition to being used as various types of foods and drinks (including foods and drinks in the present invention), they can also be used as pharmaceuticals such as cholesterol absorption inhibitors and cholesterol lowering agents.

When used as a food or drink, the peptide which is the active ingredient of the present invention can be used as it is, or can be used in combination with other foods or food ingredients according to a conventional method. The food and drink according to the present invention using the present active ingredient,
It may be in solid form (powder, granules, etc.), paste form, liquid form or suspension form, but is formulated into a health drink using sweeteners, acidulants, vitamins and various other ingredients commonly used in the production of drinks. Is preferable.

When used as a medicine, the active ingredient is administered in various forms. Examples of the dosage form include oral administration such as tablets, capsules, granules, powders and syrups. These various preparations are usually used in the technical field of pharmaceutical preparation such as excipients, binders, disintegrants, lubricants, flavoring agents, solubilizers, suspension agents, coating agents, etc. It is possible to formulate with known auxiliary agents. The amount to be used varies depending on symptoms, age, body weight, administration method, dosage form and the like, but usually, about 0.1 mg to 1,500 mg per day can be administered to an adult.

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 rats, but no acute toxicity was observed. Therefore, when used as a food or drink, there is no particular limitation on the amount of the active ingredient used for prevention, health care, or use as a food or drink, and even when used as a medicine, the above range depending on the patient. It may be appropriately used in. 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.

The embodiments of the present invention will be described in detail below.

[0015]

Example 1 Peptide L and peptide I were respectively synthesized using an automatic synthesizer. The purity of the obtained peptide is
It was confirmed to be 95% by reverse phase HPLC, respectively. With respect to these synthetic peptides, the action of lowering the serum cholesterol concentration was confirmed by an in vivo test using rats.

Cholesterol containing 20% casein
0%, using sodium cholate 0.25% added feed,
Wistar male rats (3 weeks old, initial weight 30 g) were bred for 4 days. The composition of the added feed is shown in Table 1 below.

[0017] (Table 1)                        Feed composition (g / 1.2kg)                             (%) 1C   Casein 20 279.07   Lard 5 60   Corn oil 1 12   Cellulose 560   Mineral 1 3.5 42   Vitamin 2 1 12   Sucrose 239.18   Starch 478.35   Cholesterol 1 12   Sodium cholate 0.25 3   Choline chloride 0.2 2.4   Total 1200.00   However, 1C: 20% casein + 1% cholesterol         Sucrose and starch mixed 1: 2

In the above, Mineral 1 is an AIN-76 mineral mixture, and the feed contains the following minerals in the following amounts (mg) per 1 kg of feed.

(Mineral mixture) AIN-76 mineral mixture (mg / kg): Ca, 5200
; P, 4000; K, 3600; Na, 1020; Mg, 500; Mn, 54
; Fe, 35; Cu, 6; Zn, 30; I, 0.2; Se, 0.2; Cr,
2.0; Cl, 1560; Sulfate, 1000.

In the above, vitamin 2 is an AIN-76 vitamin mixture, and the above feed contains the following vitamins in the following amounts (mg) per 1 kg of feed.

(Vitamin mixture) AIN-76 vitamin mixture (mg / kg): Thiamine-HCl, 6.0; Riboflavin, 6.0; Pyridoxine-HCl, 7.0; Nicotinic acid, 30; Pantothenate Ca, 16; Folic acid, 2.0; Biotin, 0.2; menadione, 0.05; Retinyl palmitate, 2.67 (4,000IU); Ergocalciferol, 0.025 (1,000IU); dl-α-tocopherol acetate, 100 (100 IU).

Rats were divided into 5 groups (C, β, G, L, I: 7 animals each), C group (caseintrypsin hydrolyzate), β group (β-sitosterol), G group (β). -Lactoglobulin trypsin hydrolyzate), group L (synthetic peptide L), group I (synthetic peptide I) were orally administered at a dose of 200 mg / Kg / day at 8 o'clock on the second day and the third day, respectively. .

The β-lactoglobulin trypsin hydrolyzate orally administered to the rats of group G was produced as follows, and the casein trypsin hydrolyzate of group C was also produced in the same manner.

10 kg of the β-lactoglobulin composition separated from sweet whey was dissolved in 190 kg of water, and the liquid temperature was set to 42 ° C. Add sodium hydroxide to this,
Adjust the pH to 7.5-8.0 and 0.01 NH in advance.
Trypsin dissolved in Cl solution (NOVO 4500
K) 100 g was added. After carrying out enzymatic decomposition for about 3 hours while maintaining the pH, it was immediately cooled to a pH of 6.9-
After adjusting to 7.0, the enzyme was inactivated by a plate sterilizer. By spray drying this solution, β-
About 9.0 Kg of lactoglobulin trypsin hydrolyzate was obtained.

Twenty-four hours after oral administration on the third day, the rats were sacrificed by cardiac blood sampling. Serum is 3000 rpm,
Prepared by centrifugation for 15 minutes. Serum cholesterol was quantified by an enzymatic method, specifically using a commercially available kit (Monotest cholesterol; Boehringer Mannheim Yamanouchi Co., Ltd.). As the standard, (Presetset cholesterol; Boehringer Mannheim Yamanouchi Co., Ltd.) was used. Similarly, HDL-cholesterol was measured using HDL-cholestase (Nissui Pharmaceutical Co., Ltd.). LDL + VLDL-cholesterol was calculated. The obtained results are shown in Tables 2, 3 and 4 below.
Shown in.

In the table, (a): serum cholesterol (mg / dl) (b): HDL-cholesterol (mg / dl) (c): LDL + VLDL-cholesterol (mg / d)
l) (b) / (a): Atheroge index (Atheroge
nic index), and each value is the mean ± SEM of 7 animals in one group. For statistical analysis of experimental results, Duncun's multiple range test
And Studeut's t-test were used. In the table, * p <0.
05, ** p <0.01, and *** p <0.001.

[0027] (Table 2)                              Measurement result (1)                         C β   (A) 529.31 ± 14.37d 435.38 ± 12.82c ***   (B) 42.61 ± 1.62a 52.15 ± 1.70b **   (C) 486.70 ± 26.74c 383.24 ± 24.16b *   (B) / (a) 0.08 ± 0.01a 0.12 ± 0.01b **

[0028] (Table 3)                              Measurement result (2)                         G L   (A) 441.33 ± 11.97c *** 392.49 ± 9.50b ***   (B) 50.52 ± 2.41b * 47.82 ± 1.87ab   (C) 390.81 ± 22.91b * 344.66 ± 18.14ab ***   (B) / (a) 0.12 ± 0.01b * 0.12 ± 0.01b **

[0029]

From the above results, it was confirmed that both peptides L and I have an excellent cholesterol lowering action.

[0031]

Example 2 Vitamin C 20 g, granulated sugar 50
50 g of peptide L was added to 30 g of an equivalent mixture of corn starch and lactose, and mixed well. 100 mixture
An equal portion was packed in a bag, and 100 bags of 1.5 g of stick-shaped nutritional health food for cholesterol reduction were manufactured.

[0032]

Example 3 Peptide I instead of Peptide L (50 g)
(40 g) was used, and the same procedure as in Example 2 was repeated, except that an equivalent mixture of vitamin C and citric acid (30 g) was used instead of vitamin C (20 g), and after sufficient drying. , Produced a nutritional health food for cholesterol reduction.

[0033]

Example 4 Peptide L powder 100 g, saccharide 150 g,
Honey 15g, ascorbic acid 1g, citric acid 0.5g,
Add 1 kg of water to a proper amount of fragrance and add 20 kg at 95 ℃.
Sterilize for a minute, then aseptically fill each 100 ml into a bottle,
A drink-type health drink was manufactured.

[0034]

Example 5 200 g of a 20% aqueous solution of Peptide I powder,
Tocopherol acetate 5g, thiamine nitrate 10g, nicotinic acid amide 20g, anhydrous caffeine 50g, benzoate and perfume. Add deionized water to 30L and sterilize, then aseptically fill each 30ml bottle to obtain a drug. Produced health drinks.

[0035]

Example 6 (1) Peptide L 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,
Granulated with the paste prepared from (3) (however, 7 g). To the obtained granules, (3) (however, 5 g) and (4) were added and mixed well, and the mixture was compressed by a compression tableting machine to obtain 1000 tablets each containing 50 mg of the active ingredient (1). Manufactured.

[0036]

Example 7 Tablets were produced in the same manner as in Example 6 except that Peptide I was used instead of Peptide L.

The dose may vary depending on the patient's symptoms and age, but is 0.1-1500 mg / kg / day, 1 day a day.
Administer 1 to 4 times. The peptide used in the present invention is
As described above, there is almost no problem in safety, and therefore it is safe to administer the dose over the above dose. Also,
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.

[0038]

INDUSTRIAL APPLICABILITY According to the present invention, the serum cholesterol concentration can be extremely efficiently reduced 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.

[0039]

[Sequence list]

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI A61P 3/06 A61K 37/02 (56) References JP-A-7-278010 (JP, A) JP-A-4-282400 (JP , A) JP-A-9-47229 (JP, A) International Publication 99/65326 (WO, A1) Biol. Chem. Hoppe-Seyler (1987), Vol. 368, No. 10, p. 1313-1319 Milchwissenshaft (1983), Vol. 38, No. 1, p. 18-20 Mol. Cell. Biochem. (1981), Vol. 35, No. 2, p. 103-111 (58) Fields investigated (Int. Cl. ⁷ , DB name) C07K 1/00-19/00 A23L 1/305 A23L 2/00 A61K 38/00 A61P 3/06 CA (STN ) REGISTRY (STN) BIOSIS / MEDLINE / WPID S (STN)

Claims (3)

(57) [Claims] 1. A cholesterol reduction peptide represented by the amino acid sequence of SEQ ID NO: 1. 2. A cholesterol-lowering agent comprising as an active ingredient the peptide represented by the amino acid sequence of SEQ ID NO: 1. 3. A cholesterol lowering food or drink containing a peptide represented by the amino acid sequence of SEQ ID NO: 1.