JP2001114800A - Cholesterol-lowering peptide - Google Patents

Abstract

(57) Abstract: Cholesterol-reducing peptides L and I having the following amino acid sequences: Peptide L: Leu-Asp-Ile-Gln-Lys Peptide I: Ile-Ile-Ala-Glu-Lys [Effect] A highly safe and highly effective cholesterol lowering agent or reduced food or drink is provided.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to cholesterol reduction, and more particularly to a cholesterol reduction system using a novel cholesterol-lowering peptide.

The peptide of the present invention is used not only as a cholesterol-lowering agent but also as a pharmaceutical, and also as a cholesterol-lowering beverage, food, nutritional food, functional food, food for specified health use, etc. In addition, it can be widely used for prevention of cholesterol accumulation.

[0003]

2. Description of the Related Art Sterols and their derivatives are essential constituents of living organisms. Triacylglycerols only play a role of energy storage, but cholesterol and its fatty acid esters are important constituents of cell membranes, and at the same time, have various important functions in the body such as bile acids, steroid hormones and vitamin D. This produces a derivative that performs its function. The regulation of cholesterol metabolism is important for keeping the cardiovascular system healthy, and the abnormality leads to arteriosclerosis.

In recent years, dietary fiber, soy protein,
It has been actively studied at home and abroad that various food ingredients such as unsaturated fatty acids have a favorable effect on cholesterol metabolism.

For example, Carroll and Hamilton have examined dietary proteins for plasma cholesterol levels and have shown that soy protein lowers plasma cholesterol levels as compared to casein protein (KK Carrol a).
nd RMG Hamilton, J. Food Sci., 40, 18-23 (197
Five)).

According to Japanese Patent Application Laid-Open No. 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 effect. 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 effect and an anti-atherosclerotic effect.

[0007]

SUMMARY OF THE INVENTION As described above, in view of the current state of the art in which research and development of serum cholesterol lowering agents derived from soy protein are actively conducted, the present inventors have proposed:
The development of a novel, highly effective and highly safe cholesterol-lowering agent that has never existed before and the development of a completely new type of peptide-based cholesterol-lowering agent that is not derived from soybean protein has been set as new technical subjects.

[0008]

Means for Solving the Problems The present inventors have conducted various studies to solve the above-mentioned problems, and as a result, synthesized a large number of peptides focusing on peptides, especially oligopeptides, and obtained peptides. Screening was performed. As a result, two types of peptides (peptide L and peptide I) having an excellent serum cholesterol concentration-reducing action were discovered, and as a result of further investigation, it was confirmed that they could be used as a cholesterol-lowering agent or a food-reduced food product. Thus, the present invention was completed. .

The peptides L and I according to the present invention have the amino acid sequences shown in SEQ ID NOs: 1 and 2 in 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 not been synthesized or isolated, and have a cholesterol-lowering effect. Although not yet synthesized, it can be easily synthesized and obtained by an automatic synthesizer or the like.

[0010] As will be apparent from the examples described later, these peptides have an excellent cholesterol-reducing action. Therefore, foods and drinks, foods and drinks for specified health use, health drinks, health foods, and nutritional foods utilizing this action. In addition, it can be used as various types of food and drink (in the present invention, foods include beverages), and can also be used as pharmaceuticals such as cholesterol absorption inhibitors or cholesterol lowering agents.

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

When used as a medicament, the active ingredient is administered in various forms. Examples of the administration form include oral administration by tablets, capsules, granules, powders, syrups and the like. These various preparations are commonly used in the pharmaceutical preparation technical field such as excipients, binders, disintegrants, lubricants, flavoring agents, solubilizing agents, suspending agents, coating agents, and the like, in accordance with the usual methods for the main drug. It can be formulated using known adjuvants. The amount used depends on symptoms, age, body weight, administration method, dosage form, etc., 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, shows excellent safety, and when administered orally to rats at a dose of 500 mg per day, 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, or use as a food or drink. It may be used as appropriate within. In addition, since the present active ingredient does not show any particular acute toxicity even when taken in a large amount, it may be used in an amount larger than the above range if necessary.

Hereinafter, embodiments of the present invention will be described in detail.

[0015]

Example 1 Peptide L and peptide I were synthesized using an automatic synthesizer. The purity of the obtained peptide is
Reverse phase HPLC confirmed that it was 95%, respectively. For these synthetic peptides, the effect of lowering serum cholesterol concentration was confirmed by an in vivo test using rats.

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

(Table 1) Feed composition (g / 1.2 kg) (%) 1C casein 20 279.07 lard 560 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 are mixed at a ratio of 1: 2.

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

(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 feed contains the following vitamins in the following amounts (mg) per 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; pantothenic acid Ca, 16; folic acid, 2.0; biotin , 0.2; menadione, 0.05; retinyl palmitate, 2.67 (4,000 IU); ergocalciferol, 0.025 (1,000 IU); dl-α-tocopherol acetate, 100 (100 IU).

The rats were divided into 5 groups (C, β, G, L, I: 7 rats each: 7 groups). Group C (casein trypsin hydrolyzate), group β (β-sitosterol), group G (β -Lactoglobulin trypsin hydrolyzate), Group L (synthetic peptide L) and Group I (synthetic peptide I) were orally administered at a dose of 200 mg / Kg / day at 8:00 on the second and third days, respectively. .

The β-lactoglobulin trypsin hydrolyzate orally administered to rats in Group G was produced as follows, and the casein trypsin hydrolyzate in Group C was 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 adjusted to 42 ° C. Add sodium hydroxide to this,
The pH is adjusted to 7.5 to 8.0, and 0.01 NH
Trypsin dissolved in Cl solution (NOVO 4500
K) 100 g were added. After carrying out the enzymatic degradation for about 3 hours while maintaining the pH, the mixture was immediately quenched and the pH was increased to 6.9 to 6.9.
After setting to 7.0, the enzyme was inactivated by a plate-type sterilizer. By spray drying this liquid, β-
About 9.0 kg of lactoglobulin trypsin hydrolyzate was obtained.

Twenty-four hours after the third day of oral administration, the rats were killed by cardiac bleeding. Serum is 3000rpm,
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.). The standard used was (pletisset cholesterol; Boehringer Mannheim Yamanouchi Co., Ltd.). Similarly, HDL-cholesterol was measured using HDL-cholesterase (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
nic Index), each value is the average ± SEM of 7 animals per group, and the statistical analysis of the experimental results was performed using Duncun's multiple range test
And Studeut's t-test were used. In the table, * p <0.
05, ** p <0.01, *** p <0.001.

(Table 2) Measurement results (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 **

(Table 3) Measurement results (2) GL (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 effect.

[0031]

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

[0032]

Example 3 Peptide I in place of Peptide L (50 g)
(40 g), and the same treatment as in Example 2 was repeated except that an equal mixture of vitamin C and citric acid (30 g) was used instead of vitamin C (20 g), and then dried sufficiently. Manufactured cholesterol-reducing nutritional and health foods.

[0033]

Example 4 100 g of peptide L powder, 150 g of saccharide,
Honey 15g, ascorbic acid 1g, citric acid 0.5g,
Add water to the appropriate amount of fragrance to make 1 kg, and add
Sterilize for 100 minutes, fill 100 ml aseptically into bottles,
Food and drink type health drinks were manufactured.

[0034]

Example 5 200 g of a 20% aqueous solution of peptide I powder,
5 g of tocopherol acetate, 10 g of thiamine nitrate, 20 g of nicotinamide, 50 g of anhydrous caffeine, benzoate and an appropriate amount of fragrance and deionized water were added to make up to 30 L. Manufactured healthy drinks.

[0035]

Example 6 (1) 50 g of peptide L (2) 90 g of lactose (3) 29 g of corn starch (4) 1 g of magnesium stearate (1), (2) and (3) (17 g) were mixed,
(3) Granulated together with the paste prepared from (7 g). (3) (5 g) and (4) are added to the obtained granules, and they are mixed well. This mixture is compressed by a compression tablet machine, and 1000 tablets each containing 50 mg of the active ingredient (1) are compressed. Manufactured.

[0036]

Example 7 A tablet was produced in the same manner as in Example 6, except that peptide I was used instead of peptide L.

The dosage varies depending on the patient's condition and age, but is 0.1 to 1500 mg / kg / day, once a day.
Administer 1 to 4 times. The peptide used in the present invention is
As mentioned above, there is almost no problem in safety, and therefore it is safe to administer more than the above dose. Also,
When it is used for maintenance and promotion of health, health, nutrition, etc., it is sufficient to take an amount smaller than the above-mentioned dose over a long period of time.

[0038]

According to the present invention, the serum cholesterol concentration can be extremely efficiently reduced, and the safety is extremely high. Therefore, the present invention is useful only as a prophylactic or therapeutic agent for reducing serum cholesterol concentration. No unpleasant flavor such as strong bitterness is observed, and it can be used as a food or drink for prevention or treatment for reducing cholesterol.

[0039]

[Sequence list]

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI theme coat ゛ (Reference) A61P 3/06 A61K 37/02 F-term (Reference) 4B017 LC03 LE10 LK01 LK08 LK12 LK15 LK16 LK20 LL01 LP12 4B018 LB08 LB10 LE03 LE05 MD09 MD20 MD25 MD27 MD29 ME04 4C084 AA02 AA07 BA01 BA08 BA16 CA59 DC50 MA02 MA23 MA35 MA37 MA41 MA43 MA52 ZA452 ZC332 4H045 AA10 AA30 BA13 EA01 EA27 FA34

Claims (4)

[Claims] 1. A cholesterol-lowering peptide, peptide L, represented by the amino acid sequence of SEQ ID NO: 1 in the sequence listing. 2. A cholesterol-lowering peptide, peptide I, represented by the amino acid sequence of SEQ ID NO: 2 in the sequence listing. 3. A cholesterol-lowering agent comprising peptide L and / or peptide I as an active ingredient. 4. A cholesterol-lowering food or drink containing peptide L and / or peptide I.