JPH10292000A - α-Glucosidase inhibitor and mixture thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an α-glucosidase inhibitor that can effectively inhibit the activity of α-glucosidase and is ingested as a food because of its high safety for human bodies. SOLUTION: Vegetable or animal protein is hydrolyzed, purified with ion- exchange resin and subjected to gel filtration with molecular sieve and the resultant protein with a molecular weight of 200-1,000 shows the α-glucosidase inhibitory activity. This protein still retains its α-glucosidase inhibitory activity even after it is isolated or remains in a mixture. The peptides which are isolated and have still high inhibitory activity are the following proteins of 5 kinds having amino acid sequences of formulas (1)-(5): formula (1): Ile-Ile-Ser-Ile-Gly, formula (2): Ile-Ile-Ser-Ile-Gly-Arg, formula (3): Val-Phe-Ile-Lys-Ala-Ala, formula (4): Val-Phe-Ile-Lys-Ala-Ala-Ala, and formula (5): Val-Phe-Ile-Lys-Ala.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to an α-glucosidase inhibitor comprising a peptide which inhibits the activity of α-glucosidase, or a mixture of two or more such peptides having α-glucosidase inhibitory activity.

[0002]

BACKGROUND OF THE INVENTION Starch and sucrose account for the greatest proportion of carbohydrates consumed by humans, and are said to account for 80-90% of the total carbohydrates consumed.
After the meal, starch is converted to α-amylase in saliva by α-amylase.
-Decomposed into dextrin, reaches the stomach and duodenum, is hydrolyzed to maltose and isomaltose via maltodextrin by α-amylase secreted from the pancreas, and reaches the small intestine. On the other hand, sucrose reaches the small intestine without being decomposed in the digestive organs on the way. These saccharides that have reached the small intestine are decomposed into monosaccharides, which are constituent sugars, and absorbed by α-glucosidase localized in the microcilia of the small intestine.

[0003] When starch or sucrose is ingested, it is digested and absorbed, and the blood sugar level (blood glucose concentration) sharply rises. If the blood sugar level increases in a healthy person, the secretion of insulin is stimulated, the insulin concentration in the blood increases, and the blood sugar level is adjusted. However, if too much starch or sucrose is ingested due to overeating or drinking, the glucose concentration and amount in the blood will increase, and the amount of insulin secreted will increase. When such a state continues for a long period of time, the function of the pancreas is reduced, and it is considered that this causes the onset of diabetes.

In recent years, when an α-glucosidase inhibitor is administered, the α-glucosidase inhibitor inhibits α-glucosidase localized in the microvilli of the small intestine, and suppresses a rapid increase in blood glucose level and an increase in insulin secretion after eating. Are known (for example, JP-A-52-122342, DI
ABETIC MEDICINE, 1993; 10: 688-693,134-138, Am, J. Cli
n.Nutr. 1992; 55: 318S-9S, JP-A-57-200355, Am, J. Clin. Nutr. 1992; 55: 314S-7S, JP-A-57
-59813). Among such α-glucosidase inhibitors, acarbose is used as a therapeutic drug for oral diabetes for non-insulin-dependent diabetes (abbreviation: NIDDM). However, these substances are essentially foreign substances to living organisms, and there are concerns about their safety, and strict regulations are set for the amount of use.

[0005]

As described above, the conventional α
-Since glucosidase inhibitors have a problem in terms of safety such as side effects, it is desired to develop a substance which is contained in foods usually consumed and which has high safety and which inhibits α-glucosidase. I was

Accordingly, the present invention has been made in view of the above-mentioned circumstances, and it is possible to effectively inhibit the activity of α-glucosidase, and it is highly safe for the human body, and α-glucosidase can be ingested as food. -To provide a glucosidase inhibitor.

[0007]

Means for Solving the Problems The present inventors have focused on hydrolysates of proteins derived from plants and animals which are generally ingested as foods, and those hydrolysates have an α-glucosidase inhibitory action. We examined whether or not.
As a result, they found that the hydrolyzates of these proteins have α-glucosidase inhibitory activity, and further found a special peptide having high α-glucosidase inhibitory activity obtained by purifying this hydrolyzate, and completed the present invention. did.

That is, the present invention provides a method for hydrolyzing a plant protein or an animal protein, purifying the protein with an ion-exchange resin, and filtering the resultant through a molecular sieve gel. Α-glucosidase inhibitor selected from peptides.

The present invention also provides a mixture of various peptides having the above-mentioned α-glucosidase inhibitory activity and having a molecular weight of 200 to 1,000.

[0010] The present invention also relates to a method for determining the molecular weight of peptide and α-
The relationship between glucosidase activities was examined, and a range of molecular weight showing particularly high α-glucosidase inhibitory activity, that is, a molecular weight of 2
The present invention has been found to provide α-glucosidase inhibitors in various dosage forms according to conventional formulation techniques using at least one of the peptides having the above molecular weight as an active ingredient.

The present invention also relates to any one of the α-glucosidase inhibitors selected from the peptides having α-glucosidase inhibitory activity represented by the following formulas (1) to (5).

Ile-Ile-Ser-Ile-Gly ... Formula (1) Ile-Ile-Ser-Ile-Gly-Arg ... Formula (2) Val-Phe-Ile-Lys-Ala-Ala ... Formula (3) Val -Phe-Ile-Lys-Ala-Ala-Ala Formula (4) Val-Phe-Ile-Lys-Ala Formula (5) The present invention is represented by the above formulas (1) to (5). α
A peptide mixture having α-glucosidase inhibitory activity in a state where two or more peptides having glucosidase inhibitory activity are mixed.

The peptides of formulas (1) to (5) or a mixture thereof can be produced by hydrolyzing a potato protein separated from potato with a protease. The peptides of the above formulas (1) to (5) may be produced by chemical synthesis according to a known method.

The peptide or peptide mixture having an α-glucosidase inhibitory activity of the present invention and the α-glucosidase inhibitor can be obtained by hydrolyzing a plant protein or an animal protein. Peptides with known amino acid structures can be produced by chemical synthesis, and these peptides can be said to be highly safe for the human body.

The α-glucosidase-inhibiting activity of the peptide or peptide mixture of the present invention is added to food or feed by humans or animals, or administered as an active ingredient of a medicament. Peptides or peptide mixtures having glucosidase inhibitory activity are expected to inhibit α-glucosidase in the microvilli of the small intestine of humans or animals, and to suppress a rapid rise in postprandial blood glucose and a subsequent rapid rise in insulin.

Therefore, foods or feeds to which the peptide or peptide mixture having an α-glucosidase inhibitory activity of the present invention is added may be health foods, foods for patients with diabetes, slimming foods, diets for livestock and pets, and diabetes. It is useful for preventive feeds.

[0017]

Embodiments of the present invention will be described below.

[Example 1] 1. Production of crudely purified peptide To a protein separated from potato (trade name: potato protein, manufactured by Hokuren Agricultural Cooperative Federation), 3 times the amount of a 2% aqueous sodium hydroxide solution was added, and after uniform dispersion, concentrated hydrochloric acid was added. The pH was adjusted to 7.0. Based on the protein content of the obtained dispersion, a proteinase derived from pig pancreas (trade name: Pancreatin, manufactured by Wako Pure Chemical Industries, Ltd.) and a protease derived from microorganisms (trade name: Amano P, Amano Pharmaceutical Co., Ltd.) (Manufactured by the company) in an amount of 1% by weight, and reacted at 40 ° C for 120 minutes.

The obtained reaction product was centrifuged to separate a solution fraction and a precipitate fraction. The precipitate fraction was removed, the solution fraction was decolorized with an ultrafiltration membrane, and then purified with an ion exchange resin to obtain a crude purified liquid. The crude purified liquid was desalted and concentrated by a reverse osmosis membrane, and the obtained concentrated liquid was spray-dried to obtain a powder. This powder was used as a crude peptide.

[0020] 2. Production of Purified Peptide A column for gel filtration chromatography having a column volume of 1500 CC (Toyopearl HW-4 manufactured by Tosoh Corporation)
0COURSE) and the above-mentioned 1. The purified peptide obtained in the step was purified by molecular sieving gel filtration chromatography, using pure water as the developing solution, and the flow rate was 100 ml /
Each fraction was fractionated in an amount of 15 ml to obtain hrs.

Each fraction was measured for absorbance at 280 nm and 220 nm by a known ultraviolet absorption method using a spectrophotometer. The result is shown in FIG. A powder obtained by freeze-drying a fraction obtained by combining several fractions was used as each purified peptide. The amount of each purified peptide thus obtained was measured. The results are shown in Table 1 below.

[0022]

[Table 1]

In Table 1 and FIG. In the fractions (31 to 64) with a small elution fraction, the high molecular component was fractionated, and the fraction No. with a long elution time was fractionated. In the fraction (78-140) having a large size, it is shown that low-molecular peptides are gradually fractionated.

[0024] 3. Preparation of α-Glucosidase Crude Enzyme Solution The small intestinal mucosa of a rat was collected, 0.1 M phosphate buffer (pH 7.0) containing 5 mM EDTA was added thereto, homogenized, and the precipitate was collected by centrifugation. The precipitate was suspended by adding a small amount of the same buffer, and 1% T
5 times the same buffer containing riton X-100 was added,
The mixture was stirred at 5 ° C. in a low-temperature room for 60 minutes to extract the enzyme. The precipitate was removed by centrifugation, and further dialyzed in a 0.01 M phosphate buffer (pH 7.0) to obtain a crude enzyme solution.

[0025] 4. Measurement of α-Glucosidase Inhibitory Activity A powder obtained by lyophilizing a powder obtained before purification by molecular sieve gel filtration chromatography and several fractions obtained by fractionating in the production of the purified peptide are obtained. The α-glucosidase inhibitory activity of each powder was measured as follows.

As a substrate, 20 mM sucrose 0.5 m
1 to a test tube, 0 to 0.4 ml of each powder material diluted with 0.1 M phosphate buffer (pH 7.0) to 0.2 to 2.0% by weight is added to the test tube. And 0.1 to 0.1 ml of a 0.1 M phosphate buffer (pH 7.0).
added. That is, the total amount of the 0.1 M phosphate buffer containing the above powder was 0.4 ml. The solution containing the substrate and the α-glucosidase inhibitor is
0.1 ml of 0 mM sucrose and 0.1 ml of an enzyme solution obtained by appropriately diluting a crude enzyme solution of rat intestine α-glucosidase obtained in the above step were added, and reacted at 37 ° C. for 30 minutes. Then, 2M Tris-HCl buffer (pH 7.
2) was added to stop the reaction, and the amount of glucose produced by the enzyme reaction was quantified by an enzymatic method using glucose oxidase. The enzyme inhibitory activity was defined as 100% of the reaction between the substrate alone and the crude enzyme solution, and the value obtained by subtracting the enzyme activity when the powder and the crude enzyme solution were added to the substrate from this value was defined as the inhibitory activity.

FIG. 2 shows the results of the enzyme inhibitory activity of the powder before purification by molecular sieve gel filtration chromatography. FIG.
In the horizontal axis represents the amount of powder added before purification by molecular sieve gel filtration chromatography added to the reaction solution,
The enzyme inhibitory activity is shown as a graph on the vertical axis. As shown in FIG. 2, the enzymatic activity decreased as the concentration of the powder increased. This indicates that the powder before purification by molecular sieve gel filtration chromatography has an activity of inhibiting the reaction of α-glucosidase.

The α-glucosidase inhibitory activity of the purified peptide obtained by gel filtration chromatography was measured by the above method. The results are shown in Table 2 below. Table 2 also shows the α-glucosidase inhibitory activity of the powder before purification by molecular sieve gel filtration chromatography (rough purification column).

[0029]

[Table 2]

As shown in Table 2, it can be seen that the lower the molecular weight fraction (fraction No. 84 or later), the longer the elution time in the molecular sieve gel filtration purification, the more the α-glucosidase reaction is inhibited.

In the above experiment, the fraction No. having high α-glucosidase inhibitory activity was used. 84-95, fraction no. 96-140, fraction no. 141
-163, fraction No. 164 to 188, fraction no. For one hundred ninety-nine to two hundred ten, alpha-glucosidase inhibitory rate was measured concentration IC ₅₀ of the peptide required to be 50%. The results are shown in Table 3 below.

[0032]

[Table 3]

Measurement of the molecular weight of each fraction obtained by combining several fractions was carried out using a gel filtration column (TSK manufactured by Tosoh Corporation).
gel G2500pw 7.5 × 300 mm) using a high performance liquid chromatography (LC-62 manufactured by Hitachi, Ltd.).
00) according to a conventional method. As molecular weight markers,
Serum albumin, Citrome-C, bacitracin, Gl
y-Gly-Tyr-Arg, Gly-Gly-Gly
Was used. The results are shown in Table 4 below.

[0034]

[Table 4]

According to Tables 2 and 4, the molecular weight is 200
It can be seen that α-glucosidase activity is high in １０００1000 peptides.

[Example 2] In Table 1 above, the amount of purification was particularly large, and the fraction having particularly high α-glucosidase inhibitory activity in Table 2 (fraction Nos. 96 to 1)
The powder obtained by freeze-drying 40) was dissolved in 2 ml of distilled water, and then an ODS column (COARA manufactured by Nacalai Tesque, Inc.) was used.
High performance liquid chromatography (Hitachi, Ltd. LC-62) using SMOSIL 5Cl8AR, 1 × 25 cm.
(00) to perform further fractionation.

Here, the fractionation method by the above-mentioned high performance liquid chromatography is performed by using a 0.1% TFA aqueous solution (solution A) and a 0.1% aqueous solution of TFA.
A 40% acetonitrile solution (solution B) containing 1% TFA was used. For the first 15 minutes, only the above solution A was added, and 15.1 was added.
The solution A was reduced to 100% to 0% while the solution B was increased to 0 to 100% for 4 to 45 minutes.
During the 0 minute period, only the solution B was flown to perform fractionation. At this time, the flow rate of the eluate was 2 ml / min, and the measurement was performed at UV 220 nm. 3 to 8 show charts obtained as the analysis results. According to FIG. 4, FIG. 5, FIG. 8, FIG. 6, and FIG. 7 in which the peak appeared, 39.8 minutes, 40.0 minutes, 40.3 minutes,
Peaks are shown at 40.6 minutes and 41.0 minutes.

With respect to each fraction thus fractionated, the protein amount (peptide amount) and α-glucosidase inhibitory activity were measured as follows.

Here, the measurement of the protein amount was performed as follows. That is, the measurement was performed using the known Lowry-Folin method. That is, in the second embodiment, 10 to 100
Take 0.1-0.2 ml of a sample containing μg into a test tube,
1 ml of a reagent prepared with 50 parts of a 0.1N NaOH solution containing 0.1% Na ₂ CO ₃ and 1 part of 1% sodium citrate containing 0.5% CuSO ₄ is added, and the mixture is stirred well and then cooled to room temperature.
For 1 minute and then the acid concentration of 1N Folin-Cio
Add 1 ml of calteu reagent (commercially available) quickly and add 1 ml.
After an hour, colorimetry was performed at 750 nm. Protein amount (peptide amount) from a standard curve prepared in advance with bovine serum albumin
Was calculated. The measurement of α-glucosidase inhibitory activity was performed in the same manner as in Example 1.

Further, each peak fraction in FIGS. 4 to 8 was again subjected to preparative purification by high performance liquid chromatography in the same manner as described above to purify it as a single fraction. Next, the structure of the peptides contained in the four peak fractions purified and fractionated was identified using a protein sequencer (Model 477A, manufactured by Applied Biosystems). As a result, it was confirmed that these peptides were peptides having the structural formulas shown in Formulas (1) to (5).

Ile-Ile-Ser-Ile-Gly Formula (1) Ile-Ile-Ser-Ile-Gly-Arg Formula (2) Val-Phe-Ile-Lys-Ala-Ala Formula (3) Val-Phe- Ile-Lys-Ala-Ala-Ala Formula (4) Val-Phe-Ile-Lys-Ala Formula (5) In addition, IC for each peptide of structural formulas (1) to (5)
₅₀ was measured. Table 5 shows the results.

[0042]

[Table 5]

According to Table 5, the above structural formulas (1) to (5)
It can be seen that the peptide represented by effectively inhibits α-glucosidase.

[0044]

According to the present invention, an α-glucosidase inhibitor selected from peptides having a molecular weight of 200 to 1,000 obtained by hydrolyzing the plant or animal protein of the present invention and filtering by molecular sieve gel, or a peptide mixture thereof, or The peptide mixture having the α-glucosidase inhibition composed of the peptide specified by the formulas (1) to (5) or two or more peptides of the formulas (1) to (5) effectively inhibits the α-glucosidase I do. Since the α-glucosidase inhibitor or the peptide mixture having α-glucosidase inhibitory activity of the present invention is obtained from vegetable or animal protein, it is safe when ingested or administered as food, feed, medicine or the like. It is highly useful and useful.

[Brief description of the drawings]

BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is a graph (detection UV: 280 nm and UV: 220 nm) showing the results of fractionation and purification of a potato protein protein hydrolyzate by molecular sieve gel filtration chromatography.

FIG. 2 is a graph showing the action of a protein enzyme hydrolyzate of potato protein to inhibit the digestive enzyme α-glucosidase from rat small intestine.

FIG. 3 is a chart showing the results of high-performance liquid chromatography of a powder obtained by freeze-drying a fraction having high α-glucosidase inhibitory activity (fractions Nos. 96 to 140).

FIG. 4: 39.8 in high performance liquid chromatography
5 is a chart in which a peak appears at a minute position.

FIG. 5: 40.0 in high performance liquid chromatography
5 is a chart in which a peak appears at a minute position.

FIG. 6: 40.6 in high performance liquid chromatography
5 is a chart in which a peak appears at a minute position.

FIG. 7: 41.0 in high performance liquid chromatography
5 is a chart in which a peak appears at a minute position.

FIG. 8: 40.3 in high performance liquid chromatography
5 is a chart in which a peak appears at a minute position.

────────────────────────────────────────────────── ─── Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI C12N 9/99 C12N 9/99 // A61K 38/55 ADP A61K 37/64 ADP (72) Inventor Shiomi ▲ Toku ▼ husband Sapporo, Hokkaido (72) Inventor Hideichi Onodera 7-23 Asahi Heiwa Building 1-chome, 1-chome, Shiroishi-ku, Sapporo-shi, Hokkaido

Claims (9)

[Claims] 1. A peptide having an α-glucosidase inhibitory activity and having a molecular weight of 200 to 1000, which is obtained by hydrolyzing a vegetable protein or an animal protein, purifying it with an ion-exchange resin, and performing molecular sieve gel filtration. Α-glucosidase inhibitor. 2. An α-protein having a molecular weight of 200 to 1000 obtained by hydrolyzing a vegetable protein or an animal protein, purifying it with an ion-exchange resin, and filtering through a molecular sieve gel.
A mixture of peptides having glucosidase inhibitory activity. 3. A peptide having a molecular weight of 200 to 1,000.
An α-glucosidase inhibitor comprising at least one species as an active ingredient. 4. An α-glucosidase inhibitor represented by the following formula (1). Ile-Ile-Ser-Ile-Gly ... Formula (1) 5. An α-glucosidase inhibitor represented by the following formula (2). Ile-Ile-Ser-Ile-Gly-Arg Formula (2) 6. An α-glucosidase inhibitor represented by the following formula (3). Val-Phe-Ile-Lys-Ala-Ala Formula (3) 7. An α-glucosidase inhibitor represented by the following formula (4). Val-Phe-Ile-Lys-Ala-Ala-Ala Formula (4) 8. An α-glucosidase inhibitor represented by the following formula (5). Val-Phe-Ile-Lys-Ala ... Formula (5) 9. A peptide mixture having an α-glucosidase inhibitory activity in a state where two or more peptides selected from the following formulas (1) to (5) are mixed. Ile-Ile-Ser-Ile-Gly ... Formula (1) Ile-Ile-Ser-Ile-Gly-Arg ... Formula (2) Val-Phe-Ile-Lys-Ala-Ala ... Formula (3) Val-Phe- Ile-Lys-Ala-Ala-Ala ... Formula (4) Val-Phe-Ile-Lys-Ala ... Formula (5)