JP2003252790A - Cholesterol reducer - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a cholesterol reducer containing peptides which are proven that they are excellently safe and have high efficacy by not only in vitro tests but also in vivo tests, and capable of being used as food and drink and/or pharmaceuticals for reducing the cholesterol. <P>SOLUTION: This cholesterol reducer contains a GLDIQK peptide and/or an ALPMH peptide as active ingredients, wherein the GLDIQK peptide has an amino acid sequence: Gly-Leu-Asp-Ile-Gln-Lys, and the ALPMH peptide has another amino acid sequence: Ala-Leu-Pro-Met-His. <P>COPYRIGHT: (C)2003,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 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]

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, a molecular weight of 200 obtained by hydrolyzing soybean protein with an enzyme is used.
It has been shown that ~ 1500 oligopeptides have a blood cholesterol level 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 action and an anti-atherogenic action.

[0005]

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.

[0006]

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 peptides (peptide GLDIQK and peptide ALPMH) having excellent serum cholesterol concentration-reducing action were discovered, and further studies were conducted not only in vitro tests but also in vivo tests. The present invention has been completed by confirming that the peptide can be used as a cholesterol-reducing food or drink or medical agent.

The peptides GLDIQK and A according to the invention
LPMH consists of the amino acid sequences shown in SEQ ID NOS: 1 and 2 of the sequence listing, respectively, and is shown below. Peptide GLDIQK: (in case of single letter notation) GLDI
QK; (in case of three letter notation) Gly-Leu-Asp-
Ile-Gln-Lys Peptide ALPMH: (in the case of single letter notation) ALPM
H; (in case of three letter notation) Ala-Leu-Pro-M
et-His

That is, the present invention is characterized in that a peptide having a specific amino acid sequence has an excellent cholesterol-reducing action, which is confirmed not only in an in vitro test but in an actual in vivo test. There is no report confirming the relationship between these specific sequences and the cholesterol-reducing action in an in vivo test, and the present invention is the earliest. Therefore, the present invention, which is a cholesterol-reducing food-drinking or medical agent containing at least one of these peptides as an active ingredient, is a novel one which has hitherto been unknown.

These peptides can be synthesized according to a conventional method for peptide synthesis. For example, a solid phase method in which a resin is used to add an amino acid protected by a protecting group from the C-terminal side and other conventional methods are used. Is used as appropriate. Also,
The peptide can be synthesized using an automatic synthesizer, or can be separated and fractionated from a natural product.

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 mice, 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.

Examples of the present invention will be described below.

[0015]

Example 1 Various peptide samples were added to cultured cells of Caco-2 cells (ATCC HTB-37),
In-vitro peptide that suppresses cholesterol absorption
The amino acid sequence of the peptide that suppresses cholesterol absorption was determined by searching and evaluation with ro. That is, a cholesterol absorption experiment was conducted as follows.

[0016] [4- ¹⁴ C] - cholesterol, cholesterol 0.1 mM, oleic acid 1 mM, monoolein 0.5mM, L-α-Phosphatidylcho
An emulsion containing line 0.6 mM and taurocholic acid 6.6 mM was prepared, and cholesterol absorption was measured. The culture (DMEM + 10% fetal calf serum) of Caco-2 cells cultured in a 48-well plate was absorbed and removed. The administration liquid (including the sample) prepared above is 1 mg /
Add 0.2 ml micellar solution / hole, incubator (5
% CO ₂ , 37 ° C.) (20 minutes). Thereafter, the administration liquid was absorbed and removed by an aspirator,
It was washed twice with 5 ml DMEM. The cells were peeled off by adding 0.5 ml / well of 0.1% SDS solution and transferred to a vial. 7.5 ml of Aquasol-2 (N
EN, Boston, Mass.) Was added and the amount of [ ¹⁴ C] -cholesterol absorbed was measured by a liquid scintillation counter.

As a result, a peptide having an excellent ability to suppress cholesterol absorption was successfully screened, and its amino acid sequence was also determined. Thus, the peptides GLDIQK and ALPMH (the amino acid sequences of which are shown in the sequence listing, respectively). SEQ ID NOS: 1 and 2) are obtained.

[0018]

Example 2 In Vitr of Cholesterol-Lowering Substance
o As a screening system, the peptide GLDIQK (SEQ ID NO: 1) and the peptide ALPMH (SEQ ID NO: 2) have a cholesterol absorption inhibitory effect as described above by the method using Caco-2 cultured cells developed by the present inventor. Since it was confirmed in vitro, whether or not these peptides have a serum cholesterol lowering effect in vivo by oral administration was tested as follows.

That is, using an automatic synthesizer, peptide G
LDIQK and the peptide ALPMH were each synthesized. The purity of the obtained peptide was 95% by reverse phase HPLC.
It was confirmed that each was. With respect to these synthetic peptides, the effect of lowering the serum cholesterol concentration was confirmed by an in vivo test using mice. Similarly, casein trypsin hydrolyzate (CTH), and
Trypsin hydrolyzate of β-lactoglobulin (LT
For H), an in vivo test was also performed.

LTH was prepared as follows. First, 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. Sodium hydroxide was added to adjust the pH to 7.5.
~ 8.0, trypsin (NOVO 4500K) 100 g dissolved in 0.01N HCl solution in advance
Was added. Then, the mixture was enzymatically decomposed for about 3 hours while maintaining the pH, and then immediately quenched to adjust the pH to 6.9 to 7.0.
Then, the enzyme was inactivated by a plate sterilizer. By spray-drying this solution, β-lactoglobulin trypsin hydrolyzate (LTH) about 9.0 k
g was obtained. Also, trypsin hydrolyzate of casein (C
TH) was similarly prepared.

ICR male mice having an initial body weight of about 20 g were fed with a commercially available solid diet for 3 days, and then 6 mice each were divided into groups. 20% casein with 1% cholesterol and 0.2
Casein trypsin hydrolyzate (CTH), β-lactoglobulin trypsin hydrolyzate (LTH), peptide GLDI were added to the feed supplemented with 5% sodium cholate.
The diet containing 0.3% of each of QK and peptide ALPMH was freely taken for 4 days. The diet composition is shown in Table 1 below.

[0022]

[Table 1]

In the above, the mineral is an AIN-93G mineral mixture, and the feed contains the following minerals in the following amounts (mg) per kg of the feed.

(Mineral Mixture) AIN-93G Mineral Mixture (mg / kg feed) Ca: 5000, P: 1561, K: 3600, S: 3
00, Na: 1019, Cl: 1571, Mg: 50
7, 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.

In the above, the vitamin is an AIN-93G vitamin mixture, and the feed contains the following vitamins in the following amounts (mg) per kg of the feed.

(Vitamin mixture) AIN-93G vitamin mixture (mg / kg feed): all-trans-retinyl palminate: 4000 IU, choleca
lciferol: 1000 IU, all-rac-α-tocopheryl acet
ate: 75 IU, phylloquinone: 0.75, thiamine-H
Cl: 6.36, rivoflavin: 6.0, pyridoxin-HCl:
7.29, nicotinic acid: 30, Ca-pantothnate: 1
6.31, folic acid: 2.0, cyanocobalamin: 0.
025, biotin: 0.2.

The mice were sacrificed by heart blood sampling. Serum was prepared by centrifugation at 3000 rpm for 15 minutes.
Serum cholesterol is quantified by an enzymatic method, specifically a commercially available kit (Cholesterol E-Test Wako; Wako Pure Chemical Industries, Ltd .; Peeringer Mannheim Yamanouchi Co., Ltd.)
Was measured using. The standard is (Presett cholesterol; Peeringer Maiheim Yamanouchi 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.
The results are shown in Table 3. Liver lipids can be obtained by the method of Folch et al. (Folc
h, J., Lees, M., Sloane Stanley GH. Biol. Chem., 2
26, 497-509 (1957)) and total liver lipid weight was measured gravimetrically. Liver cholesterol was measured using cholesterol E-Test Wako (Wako Pure Chemical Industries, Ltd.), and liver triacylglycerol was measured using triglyceride E-Test Wako (Wako Pure Chemical Industries, Ltd.).

[0028]

[Table 2]

[0029]

[Table 3]

In the above table, the feeding period was 4 days, and each numerical value was the average ± SEM of 6 animals per group. For statistical analysis of experimental results, Duncan's multipl was used.
e range test and Student's t-
test was used. In the table, * p <0.05, **
p <0.01, *** p <0.001, indicating that there is a significant difference between different letters (p <0.05).
Moreover, LDL-VLDL cholesterol = total cholesterol-HDL cholesterol is shown.

As is clear from the above results, the two types of peptides (GLDIQK and ALPMH) were both C
It was confirmed for the first time in the world that the arteriosclerosis index was significantly increased as compared with TH, and a serum cholesterol lowering effect was expressed (Table 2). Furthermore, it was clearly confirmed by in vivo tests that hepatic cholesterol also had a significantly lower tendency than that of CTH by ingestion of GLDIQK and a tendency of lowering with ALPMH, which was very interesting (Table 3).

[0032]

[Example 3] Vitamin C 20 g, granulated sugar 50
g, 50 g of the peptide GLDIQK was added to 30 g of an equal mixture of corn starch and lactose, and they were thoroughly mixed. The mixture was divided into 100 equal parts and packed in a bag to produce 100 bags of 1.5 g of stick-like nutritional health food for cholesterol reduction.

[0033]

Example 4 The procedure was repeated except that the peptide ALPMH (40 g) was used instead of the peptide GLDIQK (50 g), and an equivalent mixture of vitamin C and citric acid (30 g) was used instead of the vitamin C (20 g). The same treatment as in Example 3 was repeated, and after sufficiently drying, a nutritional health food for cholesterol reduction was produced.

[0034]

[Example 5] Peptide GLDIQK powder 100 g, sugar 150 g, honey 15 g, ascorbic acid 1 g, citric acid 0.5 g, and perfume proper amount with water to make 1 kg.
Sterilized at 5 ° C. for 20 minutes, and 100 ml of each bottle was aseptically filled into bottles to prepare food and drink type health drinks.

[0035]

Example 6 20% aqueous solution of peptide ALPMH powder 2
00g, tocopherol acetate 5g, thiamine nitrate 10
g, nicotinamide 20 g, anhydrous caffeine 50 g,
Deionized water was added to an appropriate amount of benzoate and perfume to make 30 L, and after sterilizing, 30 ml each was aseptically filled into pins,
Made a health drink as a medicine.

[0036]

Example 7 (1) Peptide GLDIQK 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.

[0037]

Example 8 Peptide A instead of Peptide GLDIQK
Tablets were prepared in the same manner as in Example 7, except that LPMH was used.

The dose varies 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.

[0039]

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.

[0040]

[Sequence list]                 SEQUENCE LISTING <110> Meiji Dairies Corporation <120> Hypocholesterolemic Agent <130> 6526 <141> 2002-3-4 <160> 2 <210> 1 <211> 6 <212> PRT <213> Artificial Sequence <400> 1 Gly Leu Asp Ile Gln Lys                   Five <210> 2 <211> 5 <212> PRT <213> Artificial Sequence <400> 2 Ala Leu Pro Met His                   Five

Front page continuation (51) Int.Cl. ⁷ identification code FI theme code (reference) // C07K 7/06 ZNA A23L 2/00 F (72) Inventor Yoshitaka Kanamaru 5-63 Ozaki Minamimachi, Kakamigahara-shi, Gifu F-term (Reference) 4B017 LC03 LE10 LK15 LL09 4B018 LB08 LE01 LE05 MD20 ME14 4C084 AA02 BA01 BA08 BA16 BA17 BA23 MA01 NA14 ZC332 4H045 AA10 AA20 BA13 BA14 EA23 FA10

Claims (2)

[Claims] 1. A cholesterol-reducing food-drinking or medical agent comprising a peptide GLDIQK represented by the amino acid sequence of SEQ ID NO: 1 in the sequence listing as an active ingredient. 2. A cholesterol-reducing food-drinking or medical agent comprising the peptide ALPMH represented by the amino acid sequence of SEQ ID NO: 2 in the sequence listing as an active ingredient.