JP2021014419A - Pai-1 inhibitors and compositions containing mackerel peptides as effective components - Google Patents

Abstract

To provide highly safe PAI-1 inhibitors and compositions by identifying components derived from mackerel that suppress PAI-1 activity.SOLUTION: The amount of peptides in an extract prepared from mackerel meat is measured, a diluent is prepared to a predetermined concentration, and the PAI-1 activity-inhibiting action is measured. With regard to a diluent exhibiting effective PAI-1 activity-inhibiting activity, fractionation based on molecular weight, polarity and hydrophilicity are carried out using high performance liquid chromatography (HPLC) analysis, and the PAI-1 activity-inhibiting action is measured on a fraction indicating a peak. Peptides as candidates for effective components are narrowed down, and two dipeptides (Gly-Lys, Pro-Lys) are identified as the effective components.SELECTED DRAWING: Figure 1

Description

The present invention is a PAI-1 inhibitor and composition containing a mackerel-derived peptide having an action of inhibiting the activity of plasminogen activator inhibitor type 1 (hereinafter referred to as "PAI-1") as an active ingredient. Regarding.

In the blood vessel, the action of coagulating blood is maintained to stop bleeding at the time of blood trauma, but at the same time, it is necessary to maintain fluidity without obstructing the blood vessel by an excessive thrombus (blood coagulation mass). In order to maintain blood fluidity, the activities of two systems, called blood coagulation system and fibrinolytic system, which dissolve thrombi in blood vessels, are regulated so as not to be biased.

PAI-1 is one of the factors contained in the fibrinolytic system, and is known to play a role as a brake that suppresses lytic enzymes for thrombi so that the fibrinolytic system does not act excessively. Therefore, a substance that inhibits PAI-1 activity has been developed and studied as a thrombolytic drug that increases fibrinolytic activity in the treatment of cardiovascular diseases such as myocardial infarction and cerebral infarction. In the development of PAI-1 inhibitors, the relationship between PAI-1 and cancer stem cells, PAI-1 and multiple sclerosis, PAI-1 and aging has been studied, and it has been studied for a wide range of diseases. Has been studied for clinical application (see Non-Patent Document 1).

The present inventor investigated the effect of a water extract component solution (extract) from the fermented mackerel products Heshiko and Nare Zushi on PAI-1 activity in rats, and the extract directly exerted PAI-1 activity in vitro. It is reported that the inhibitory activity was not lost after digestion of the extract with digestive enzymes (see Non-Patent Document 2).

Regarding such substances that affect PAI-1 activity, for example, Patent Document 1 states that the first region consisting of 9 or less amino acid residues including the glycine residue at position 374 in PAI-1 or PAI-1. A PAI-1 inhibitor consisting of a substance capable of binding to a second region including a region from asparagine at position 31 to serine at position 35 is disclosed. Further, Patent Document 2 describes the amino acid sequence (R1-Arg-Met-Ala-Pro-Glu-Glu-Ile-Ile-Met) as a peptide derived from human plasminogen activator inhibitor 1 (PAI-1). An isolated peptide having −Asp-Arg-Pro-Phe-Leu-Phe-Val-Val-Arg-R2), where R1 is selected from the group consisting of hydrogen, acetyl, alkyl, and amino protecting groups. R2 is disclosed as an isolated peptide selected from the group consisting of carboxyls, amides, alcohols, esters, and carboxyl protecting groups.

Japanese Unexamined Patent Publication No. 2015-147744 Japanese Patent No. 5421257

Takashi Dan, "PAI-1 Inhibitor, Its Potential and Clinical Application," Journal of the Japanese Society of Thrombosis and Hemostasis, 2015, 26 (3), p. 310-317 Koji ITO, "Inhibitory effect of water extractive components from heshiko and narezushi on plasma PAI-1 activity in rats", Journal of the Japanese Society of Thrombosis and Hemostasis, 2016, 27 (3), p. 349-357

Based on the above-mentioned findings so far, the present inventors have been conducting research to identify a component that suppresses PAI-1 activity by focusing on mackerel fish meat. By identifying the component that suppresses PAI-1 activity from such foods, a PAI-1 inhibitor having high efficacy and safety can be obtained.

Therefore, an object of the present invention is to provide a highly safe PAI-1 inhibitor and composition by identifying a component that suppresses PAI-1 activity derived from mackerel.

The PAI-1 inhibitor according to the present invention contains at least one of the mackerel-derived peptides Gly-Lys and Pro-Lys as an active ingredient.

The composition for inhibiting PAI-1 according to the present invention contains at least one of the mackerel-derived peptides Gly-Lys and Pro-Lys as an active ingredient.

By identifying the mackerel-derived peptides Gly-Lys and Pro-Lys as components that suppress the PAI-1 activity derived from mackerel, highly safe PAI-1 containing at least one of these peptides as an active ingredient. Inhibitors can be obtained.

It is a graph which shows the peptide concentration when the extract was hydrolyzed for up to 9 hours. It is a graph which shows the change of the PAI-1 activity inhibitory action by a hydrolyzate. It is a graph which shows the peptide distribution by RPC of the GPC fraction about the chub mackerel hydrolyzate. It is a graph which shows the PAI-1 activity inhibitory action in the GPC fraction of a hydrolyzate. It is a chromatograph of the fraction by the hydrophilic fraction of the chub mackerel hydrolyzate. It is a chromatograph of the fraction by the hydrophilic fraction of Atlantic mackerel hydrolyzate. It is a graph which shows the residual PAI-1 activity of plasma by a peptide. It is a graph which shows the residual PAI-1 activity by the concentration change of a peptide.

Hereinafter, embodiments according to the present invention will be described in detail. As described in Non-Patent Document 2 described above, the hot water extract component solution (extract) from the fermented mackerel products Heshiko and Nare Zushi has the effect of inhibiting PAI-1 activity. It has been confirmed that. Therefore, fish meat collected from mackerel was artificially hydrolyzed with an enzyme to prepare an extract containing a peptide. Here, examples of mackerel include fish species such as chub mackerel, blue mackerel, and Atlantic mackerel. The amount of peptide in the obtained extract was measured, prepared into a diluted solution having a predetermined concentration, and the PAI-1 activity inhibitory effect was measured. Then, a molecular weight fraction, a polar fraction, and a hydrophilic fraction are fractionated by an analysis method using a high performance liquid chromatograph (HPLC) on a diluted solution showing effective PAI-1 inhibitory activity, and the fraction showing a peak is obtained. The PAI-1 activity inhibitory effect was measured again. In this way, the peptides having a molecular weight fraction as an active ingredient were narrowed down, and two types of dipeptides (Gly-Lys and Pro-Lys) were identified as active ingredients.

When animal experiments were conducted in which the identified dipeptides were synthesized and continuously administered to rats to verify their PAI-1 inhibitory ability, a clear difference in activity value was observed between the control group and the dipeptide-administered group. From, we were able to confirm its effectiveness. In addition, when the direct inhibitory ability of PAI-1 activity in vitro was verified using the synthesized dipeptide, a clear inhibitory ability could be confirmed.

Based on the above verification results, a PAI-1 inhibitor containing at least one of the mackerel-derived peptides Gly-Lys and Pro-Lys as an active ingredient can be obtained, and the mackerel-derived peptide Gly A composition for inhibiting PAI-1 containing at least one of -Lys and Pro-Lys as an active ingredient can be obtained.

Hereinafter, examples will be described.

[Example 1]
<Sample preparation>
Distilled water (1.5 times the weight ratio) is added to shredded fish meat (including bloody meat; pH is approximately 5.5 to 6) collected from chub mackerel and Atlantic mackerel, and water is adjusted to pH 7.5 to 8. A stock solution is prepared by adding a solution of sodium oxide. In order to eliminate the influence of visceral enzymes, only fish meat was used for fish meat, but in mass production, it is considered that there is no problem even if the whole fish body is processed.

Six types of commercially available proteases (papain, thermolysin, alcalase 2.4L, Amano A, Amano M, Amano N) are suspended in distilled water adjusted to pH 8 in advance, and each protease is added to the stock solution in an amount of 1% by weight. Then, it was hydrolyzed at 37 ° C. for up to 24 hours. After sampling over time and adjusting the pH to 5 to greatly reduce the activity, the reaction was stopped by further heating in boiling water at 100 ° C. for 15 minutes, and the hydrolyzate was filtered through a filter (pore size 0.45 μm). The solid matter was removed by filtration to prepare a sample solution.

<Measurement of peptide concentration by Lowry method>
The following solutions required for the Lowry method were prepared.
Solution A: Solution of 20 g of anhydrous sodium carbonate dissolved in 0.1 N sodium hydroxide solution (1 L) Solution B: 0.5 g of copper sulfate pentahydrate and 1 g of tartrate (sodium salt or potassium salt) After adjusting the pH to 6.3 by dissolving in distilled water, solution C solution with a final volume of 100 mL: solution D solution in which 1 mL of solution B is mixed with 50 mL of solution A: commercially available phenol reagent (Folin-Ciocalteu reagent) ) Was diluted 2-fold with distilled water. To measure the solution, add 5.0 mL of C solution to 1.0 mL of the sample solution, stir well, and then let stand at room temperature for 10 minutes or more, and 0.5 mL of D solution. After 30 minutes, the absorbance at 750 nm was measured with an ultraviolet visible spectrophotometer (manufactured by Shimadzu Corporation; UV-1240). The peptide concentration was quantitatively measured by comparing the obtained measurement data with a calibration curve prepared with a standard protein.

<Changes in peptide concentration due to hydrolysis>
In the fresh fish meat used as a raw material in the fish species of chub mackerel and Atlantic mackerel, the amount of water-soluble peptides was small, and when left at 37 ° C. for 24 hours, it increased about 4-fold in chub mackerel and Atlantic mackerel. As a result of adding 6 kinds of commercially available proteases to the fish meat of chub mackerel and hydrolyzing them, the water-soluble peptide increased significantly after 1 hour and gradually increased up to 9 hours. FIG. 1 is a graph showing the peptide concentration when hydrolyzed for up to 9 hours. With any of the proteases, the amount of water-soluble peptide reached almost the maximum amount by the hydrolysis time of 9 hours, and the amount of peptide tended to decrease slightly when the hydrolysis time was 24 hours. From these results, it was confirmed that the hydrolysis time is preferably 9 hours.

<Measurement of PAI-1 activity inhibitory effect>
PAI-1 activity was measured by the following measurement procedure using a commercially available measurement kit (manufactured by ABCAM; PAI-1 (SERPINE1) Rat ELISA Kit).
-Prepare a PAI-1 standard solution with an activity amount of 10 ng / mL using a 3% solution prepared by dissolving commercially available bovine serum albumin in TBS buffer according to the procedure of the measurement kit.-Sample solution whose peptide concentration was measured ( (Hydration 9 hours) is diluted to prepare a sample solution with a concentration of 10 ng / mL. ・ Equal amounts of the prepared sample solution and PAI-1 standard solution are mixed to prepare a treatment solution with an active amount of 5 ng / mL, and the temperature at room temperature After stirring for 10 minutes with, measure the amount of PAI-1 activity with a measurement kit.

<Changes in PAI-1 inhibitory activity>
FIG. 2 shows the measurement results of the amount of PAI-1 activity. As shown in FIG. 2, the amount of PAI-1 activity was much lower than 5 ng / mL in all the hydrolyzed samples, and an inhibitory effect on the activity was observed. In addition, an inhibitory effect on PAI-1 activity was also observed in the sample solution for 1 hour of hydrolysis, and no clear difference was observed depending on the type of enzyme and the hydrolysis time. These measurement results suggest that hydrolysis of fish meat produces peptides with PAI-1 inhibitory activity.

<Peptide fractionation in sample solution>
An 8020 HPLC system manufactured by Tosoh Corporation was used as the liquid chromatograph device.
(Molecular Weight Fraction: Gel Permeation Chromatography (GPC))
Using a commercially available column (manufactured by GE Healthcare Japan Co., Ltd .; Superdex 30 Increase), a 30% acetonitrile (ACN) solution containing 0.1% trifluoroacetic acid (TFA) at room temperature (25 ° C.) is used as an eluent. Peptides in the sample solution were separated using. The flow rate was 0.5 mL / min, the absorbance at 214 nm was monitored, and the eluate was collected every minute.
(Differentiation by polarity: reverse phase chromatography (RPC))
After the fraction by the molecular weight fraction was evaporated to dryness, it was redissolved in distilled water and reverse phase HPLC was performed as follows.
-Use ODS-80Ts (manufactured by Tosoh Corporation; QA grade, φ4.6 x 250 mm) for the column.-Liquid A (containing 1% ACN solution containing 0.1% TFA and solution B (containing 0.1% TFA). Prepare a 95% ACN solution) ・ Elute the sample by changing the ratio of solution A and solution B according to a predetermined two-component gradient program while monitoring the absorbance at 214 nm and 230 nm at a flow rate of 1.0 mL / min.

<Peptide separation and PAI-1 activity inhibitory effect>
A comparison of the molecular weight distributions of peptides by GPC and RPC according to the type of enzyme in a sample of 9 hours of hydrolysis revealed that there was a clear peptide in the hydrolyzate of Masaba by Amano N, as shown in FIG. confirmed. The existence of peptides has been confirmed for hydrolysates other than Amano N, but from the viewpoint of the amount of peptides in the hydrolysates and the possibility of low-molecular-weight peptides based on the molecular weight fraction, the hydrolysates by Amano N will be described later. Analysis was carried out.

Further, when the PAI-1 activity-suppressing action was measured for the GPC fractions (Fr34 to Fr37; molecular weight 200 to 450), as shown in FIG. 4, the PAI-1 activity-suppressing action was suppressed in the chub mackerel and Atlantic mackerel fractions. The effect was confirmed, but not clearly confirmed with herring.

From the above measurement results, it was confirmed that the peptide obtained by hydrolyzing the extract of mackerel contained a peptide having a PAI-1 activity inhibitory effect.

[Example 2]
Next, an analysis was performed to identify a peptide having a PAI-1 activity inhibitory effect from the hydrolyzate of mackerel extract.

<Sample preparation>
A sample solution was prepared in the same manner as in Example 1 using chub mackerel and Atlantic mackerel. Amano N was used as a protease and hydrolyzed for 9 hours.

<Peptide fractionation and identification in sample solution>
In the same manner as in Example 1, the sample solution was fractionated by molecular weight. From the verification results obtained in Example 1, the GPC fraction is narrowed down to a molecular weight of 200 to 450 for fractionation, but since it is considered that there are many peaks with strong hydrophilicity in this GPC fraction. , The following hydrophilic fractions were performed.
-Use ZIC-HILIC (Merck; SeQuant, φ4.6 x 150 mm) for the column.-Prepare an eluent (70% ACN solution containing 30% ammonium formate; pH 6.2) -Flow flow rate 1.0 mL / Elute the sample by changing the proportion of water eluent according to a predetermined two-component gradient program while monitoring the absorbance at 230 nm.

Due to the hydrophilic fraction, for chub mackerel, 2 fractions from Fr37-4 as shown in FIG. 5, and for Atlantic mackerel, 3 fractions from Fr37-4 as shown in FIG. Was done. Since the eluent contains ammonium acetate, the fraction that appears after desalting in advance and performing RPC fractionation is subjected to molecular weight analysis using a liquid chromatograph mass spectrometer (API2000 manufactured by Applied Biosystems). It was.

As a result of molecular weight analysis, it was confirmed that it was a set of multiple components, so amino acid sequence analysis (using ABI's procedure491HT) was performed on the peak fraction, and amino acid composition analysis after hydrochloric acid hydrolysis (JEOL Ltd.) JLC-500 / V2 manufactured by JLC-500 / V2 was used). As a result, two types of dipeptides (Gly-Lys and Pro-Lys) were identified from both chub mackerel and Atlantic mackerel.

[Example 3]
<Verification of PAI-1 inhibitory effect by animal experiments>
Next, in order to investigate the PAI-1 inhibitory effect of these dipeptides, the identified peptides were synthesized and continuously administered to lipid-loaded Wistar rats (manufactured by Nippon SLC Co., Ltd.), and the same as in Example 1. The PAI-1 activity inhibitory effect was measured to examine the PAI-1 inhibitory ability.

First, in the animal test, 9-week-old Wistar rats were purchased and commercial feed (CE-2, CE-2,) under the environment of room temperature 23 ± 1 ° C, humidity 55 ± 5%, and light-dark cycle 12 hours (light period 8/20 o'clock). After feeding (manufactured by Japan Claire Co., Ltd.) and pre-breeding for 2 weeks, switching to the experimental lipid-loaded diet (QuickFat, manufactured by Japan Claire Co., Ltd.) and pre-breeding for another 2 days, the rats were divided into 3 groups. The administration test was started separately.

The peptide sample for administration was prepared by first dissolving the peptide in distilled water so as to have a concentration of 1 mg / mL. The amount of water consumed and the body weight were measured periodically from the preliminary breeding to just before the administration, and the peptide dose was appropriately diluted from the amount of water consumed per day so as to be 10 mg / kg. After completion of administration for 10 days, the abdomen was opened and blood was collected, a 3.2% sodium citrate solution was added so as to contain 10%, and plasma was prepared by centrifugation. The same PAI-1 activity inhibitory effect as in Example 1 was measured on the obtained plasma. The measurement results are shown in FIG.

In FIG. 7, a decrease in PAI-1 activity was clearly observed in the peptide-administered group as compared with the control group, and the PAI-1 inhibitory effect of the peptide was confirmed.

<Verification of direct inhibitory ability in vitro>
All of the samples used this time are dipeptides, and it is known that if they are of this size, they will be absorbed as they are in the intestinal tract. Therefore, the ability to directly inhibit PAI-1 activity was examined.

First, the mixture was mixed so that the peptide content was 0.05 to 50 ng / mL with respect to the PAI-1 activity amount of 5 ng / mL, and the mixture was stirred at room temperature for 10 minutes. Next, the residual PAI-1 activity of the obtained treatment liquid was measured using the measurement kit used in Example 1.

FIG. 8 is a graph showing the measurement results. In FIG. 8, it was shown that the residual activity value was 4 ng / mL or less even at a concentration of 2 ng / mL or less, and a clear inhibitory ability was confirmed.

It was confirmed that the present invention has an effect of suppressing thrombus formation by reducing PAI-1 activity in plasma and inducing enhancement of the fibrinolytic system. However, the binding between the peptide and PAI-1 was not so strong, and the concentration dependence was not clearly observed. Therefore, it is considered that the action is not as strong as that of the drug. Therefore, although its efficacy as a therapeutic agent at the onset of thrombosis is unknown, it is expected to act as a preventive measure for vascular infarction by continuously ingesting it for a long period of time.

Claims (3)

A PAI-1 inhibitor containing at least one of Gly-Lys and Pro-Lys, which are peptides derived from mackerel, as an active ingredient. A composition for inhibiting PAI-1 containing at least one of Gly-Lys and Pro-Lys, which are peptides derived from mackerel, as an active ingredient. The composition for inhibiting PAI-1 according to claim 2, which is a composition for food.