JP6792931B1 - Strategies for avoiding binding of catechins to an unspecified number of proteins and preparation of pilot samples - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide petitide and a method thereof for suppressing non-specific binding of a protein to catechins to maintain the biological activity of the catechins, and a method for measuring the protein binding property of the catechins. A composition containing peptides and catechins. A method of suppressing non-specific binding of a protein to catechins using a peptide. A method for measuring the degree of protein binding to catechins based on the shape of a precipitate formed by methyl cellulose. [Selection diagram] Fig. 1

Description

The present invention relates to a method for inhibiting non-specific binding of a protein to catechins by making full use of a low molecular weight peptide or an oligopeptide having a large molecular weight for the purpose of suppressing non-specific binding of a protein to catechins. More specifically, the present invention relates to peptides and methods for inhibiting the non-specific binding of proteins to catechins by peptides and maintaining the biological activity of catechins.

It has been reported that green tea catechins have various physiological activities, and many products using them have been developed. Recently, derivatives with further enhanced biological activity have been developed by adding chemical modifications, which seems to open up future prospects.

On the other hand, when the expression of such physiological biological activity is examined in detail, most of them are limited to the activity test examination in vitro, or in the case of antibacterial activity, the range of the culture system is limited. In only a few papers, there is a report at the end of the presentation that "the activity could not be exhibited in vivo, that is, at the animal level including mice". In addition, catechins have a low rate of intestinal absorption, and blood and tissue catechin concentrations do not indicate in vitro activity levels, a few reports indicate that catechins have a bioactive effect in vitro. It is hard to say that it reflects the true appearance of catechin, being drowned out in articles and reports that emphasize. It is considered that the background of such a gap is solely due to the strong unspecified number of protein-binding chemical properties of catechin.

Peptides that bind to catechin have also been found (for example, Patent Document 1), but it is said that the binding of these peptides to catechin suppresses the activity originally possessed by catechin (cell growth inhibitory activity, etc.). There was a problem.

Therefore, there has been a demand for a composition or method for suppressing non-specific binding of a protein to catechins and a catechin composition that can be administered while maintaining the biological activity of catechins.

Patent No. 4348436

Provided are a peptide that maintains the biological activity of catechins even after suppressing non-specific binding of an unspecified number of proteins to catechins, a method thereof, and a simple and new method for measuring protein binding of catechins.

(1) A composition containing peptides and catechins.
(2) The composition of (1), wherein the peptide is carnosine.
(3) A method of suppressing non-specific binding of a protein to catechins using a peptide.
(4) The method of (3), characterized in that catechins maintain biological activity.
(5) The method according to any one of (3) or (4), wherein the peptide is composed of 2 to 3 amino acids.
(6) The method according to any one of (3) or (4), wherein the peptide is carnosine, glutathione and / or hepatocyte growth factor.

(7) A method for measuring the degree of binding of an unspecified number of proteins to catechins based on the shape of a precipitate formed by methyl cellulose as a result of the reaction between catechins and methyl cellulose.
(8) A peptide consisting of 2 to 3 amino acids that binds to catechins and does not inhibit the biological activity of catechins.

According to the present invention, there are provided peptides and methods capable of suppressing non-specific protein binding of catechins and maintaining the bioactivity of catechins.

FIG. 1 is a diagram showing the shape of the precipitate.

Catechins have various actions, such as antioxidant action, antitumor action, carcinogenesis inhibitory action, blood pressure increase inhibitory action, antibacterial action, antiviral action, anticariogenic action, antiallergic action, deodorant action, and lipid metabolism. Physiological activities such as improving action are known.

However, when catechins are administered in vivo, there is a problem that proteins and the like in the body bind non-specifically and the physiological activity originally possessed by catechins is significantly suppressed. Therefore, as a result of diligent research, the inventors have found a method and a peptide capable of suppressing a decrease in physiological activity due to non-specific binding of a protein or the like to catechins and maintaining / demonstrating the original physiological activity of catechins.

That is, in the present invention, by binding a peptide such as carnosine to catechins, non-specific binding of a protein to catechins is transiently suppressed, and the catechin-peptide exerts the original physiological activity of catechins. The complex and method are provided.

In the present invention, "catechins" refers to flavonoids having flavan-3-ol as a basic skeleton. Examples of molecules contained in catechins include epigallocatechin-3-O-gallate (EGCG), epigallocatechin (EGC), epicatechin-3-O-gallate (ECG), epicatechin (EC), and epigallocatechin. Galocatechin-3-O (-3-O-methyl) gallate (EGCG3 "Me), epigallocatechin-3-O- (4-O-methyl) gallate (EGCG4" Me), thearubigin (TF1), theaflavin-3 -Gallocatechin (TF2A), theaflabin-3'gallate (TF2B), theaflabin-3-3 / jigallocatechin (TF3), theacinencins, thearubigins, etc., but are not limited to these.

In the present invention, "the original physiological activity of catechins" means, for example, anticancer, suppression of plasma cholesterol elevation, suppression of blood pressure elevation, suppression of platelet aggregation, suppression of blood glucose elevation, prevention of dementia, anti-ulcer, anti-inflammatory, antiallergic. It refers to, but is not limited to, antibacterial, anti-worm tooth, anti-virus, detoxification, improvement of intestinal flora, etc.

The "peptide" in the present invention refers to a peptide in which two or more amino acids are bound, preferably 2 to 30, 2 to 25, 2 to 20, 2 to 15, 2 to 10, 2 to 9, 2 to 8, It may be a peptide in which 2-7, 2-6, 2-5, 2-4 amino acids are bound.

The "composition" in the present invention may be a food composition or a pharmaceutical composition. The food composition may be a solid food, or may be a liquid or gel-like food, and is not limited thereto. The pharmaceutical composition may be, for example, powders, tablets, capsules, liquids and the like, and is not limited thereto.

In the present invention, by binding a peptide to such catechins, non-specific binding of a protein to the catechins can be suppressed and the physiological activity inherent in the catechins can be exhibited. The peptide of Patent Document 1 inhibits the cell growth inhibitory action inherent in catechin by binding to catechin, but the peptide of the present invention is characterized in that it does not inhibit the original action of catechin even when bound to catechin. ..

As used herein, "cytopathic effect" refers to the effect of an additive on cells when a passaged cell is seeded at a predetermined concentration and a cultured monosheet is added with a compound to be tested or infected with a pathogen. If there is, it means the case where the following shape changes occur. (1) The luster characteristic of living cells is lost (2) Atypical granules appear inside the cells, (3) The shape of the cells themselves becomes circular (4) As a result, the adhesion between cells is impaired. If (5) further progresses, the monosheet will be reticulated with large gaps due to the detachment of dead cells from the bottom of the container, and (7) finally, part or all of the bottom of the container will be exposed. It refers to a series of processes leading up to.

In the present invention, the protein binding property to catechins was measured by focusing on the fact that the amount and morphology of precipitation due to the addition of methyl cellulose to catechins have a negative quantitative correlation with the protein binding to catechins. .. That is, when the amount of protein bound to catechins is small, a large precipitate is formed by adding methylcellulose, and when the amount of protein bound to catechins is large, no precipitate is formed or is small. The protein binding to catechins was examined. It is considered that this is because when the protein binding site of catechins is masked by some substance, methyl cellulose cannot bind to the masked catechins, resulting in less precipitation.

As a result of searching for peptides containing multiple carnosine by the above concept, when carnosine is added to catechins, precipitation by methyl cellulose is small, and by adding carnosine, the protein binding site of catechins can be masked. It was considered.

Addition of glutathione and hepatocyte growth factor (LCGF) to polyphenon 60 also suppressed the formation of methylcellulose precipitates, albeit weaker than carnosine (Table 3). Therefore, it was considered that the peptide consisting of 2 to 3 amino acids suppressed the non-specific binding of the protein to catechins.

In addition, carnosine's non-specific binding inhibitory activity of proteins to catechins was observed not only in polyphenon 60 but also in EGCG (Table 4). This suggests that carnosine's non-specific binding inhibitory activity of this protein is similar to other catechins.

The reason can be explained by considering that peptides such as carnosine block hydroxyl groups of catechins. This is because catechins have flavan-3-ol as the basic skeleton and have a large number of hydroxyl groups.

The present invention also includes peptides consisting of several, more preferably 2-3 amino acids, which bind to catechins and do not inhibit the biological activity of catechins. Such peptides are easy to synthesize, and those skilled in the art can easily obtain such peptides by observing precipitation formation using methyl cellulose described in the present specification and examining the cytopathic activity described below. Because it can be done.

Next, when catechins mixed with carnosine (carnosine-catechin complex) were added to the cells, cell degeneration occurred, and it was found that the catechins bound to carnosine retained the cell degenerative activity. From this, it was considered that by adding a peptide such as carnosine to catechins, non-specific protein binding can be suppressed without losing the physiological activity of catechins.

The carnosine-catechin complex acquired a stronger cell degenerative effect than Polyphenon 60 alone. This reveals that as a result of the formation of the complex, not only the protein binding (Table 3) was lost, but rather the other biological activities such as cell degenerative action were retained without being impaired. There is. This fact was reproduced not only in the crude product polyphenon60, but also in the pure catechin EGCG.

(Example 1: Establishment of a detection method for detecting protein binding of catechin)
As a catechin source, polyhenon 60 containing a high proportion of green tea catechin was used. We also found that commercially available methylcellulose (MC) reacts strongly with polyhenon 60 and its products can be easily identified under a microscope. It was also confirmed that the degree of protein binding decreased in inverse proportion to this reaction. By scoring the morphology and amount of the product, it became possible to display the protein binding property in an inversely correlated relationship.

(Table 1) Protein binding by Polyphenon 60 and quantification of binding degree by MC Calf serum (FCS) was used as the protein source. FCS was mixed at a predetermined ratio with Polyphenon 60 dissolved in 1% of HBSS (Hanks) solution, and reacted at room temperature for 4 hours. Then, the total amount was adjusted to 200 μl with physiological saline (0.9% (w / v) sodium chloride solution), and a trace amount of 1% methylcellulose (MC, 25 μl) was mixed and observed under a microscope. The degree of protein binding was scored from 1 to 4 for the clothization and stringation of MC.

(Note): As a result, Polyphenon 60 alone turned from a colorless and transparent liquid gel as a result of a strong agglutination reaction with MC, resulting in a wide range of string or cloth-like formation. On the other hand, the shape and appearance rate of MC precipitates decreased in inverse correlation with increasing the mixing ratio of FCS to polyphenon 60. This indicates that MC can be used as a sensor to measure the degree of protein binding of Polyphenon 60.

In addition, the green color of the Polyphenon 60 solution changed in color after the reaction time and turned brown, which proved that the reaction proceeded.

The activity of the block at the protein binding site of FCS catechin as a protein was not high. Blocking 10% polyphenon 60 almost perfectly required 3-4 times 30% -40% FCS.

(Example 2: Examining the protein binding block of green tea catechin (polyphenon 60) and EGCG with amino acids or peptides)
From the results in Table 1, the actual inhibition of protein binding was examined with amino acids and peptides using MC as a detection agent (sensor).
Table 2. Examination of protein binding block of amino acid Polyphenon 60

Amino acids were adjusted to 100 mM with saline as shown in the table. The reaction products were scored by reacting 1% polyphenon 60 with amino acids at varying concentrations (room temperature 3 hours) and adding 25 μl of 1% MC (saline).

(Note): Even if the reaction was carried out by increasing the proportion of amino acids, no reaction product below the reaction product (morphology) level of Control (physiological saline) at the same concentration was confirmed. Therefore, the amino acid Polyphenon 60 (green tea) was not confirmed. The protein-binding block of catechin) was determined to be incomplete.

(Example 3: Examination of protein binding block of polyphenon 60 by peptide)
As the peptide, commercially available carnosine and glutathione were used as dipeptides, and hepatocyte growth factor (LCGF) was used as a tripeptide. Peptides were adjusted to 100 mM or 50 mM with saline as shown in the table. 1% polyphenon 60 was reacted with peptides at varying concentrations (aged at room temperature for 3 hours), the total volume was increased to 0.2 ml, and 25 μl of 1% MC was added to mirror and score the reaction product. did.

(Note): Strong nonspecific protein-binding block activity of catechin was observed in Carnosine. The rationale for this is that in the Table 2 experiment in which a 100 mM amino acid solution was added to 1% polyphenon 60 (the molecular weight cannot be calculated because it is not a pure simple substance), the reaction was strong at 10: 1 (polyphenon 60 0.1%). In the case of Carnosine, almost nothing was precipitated even though the same 100 mM was reacted, while a large number of membranous agglomerates and many curtain fragments were precipitated (not blocked). Based on. When Glutathione was reacted at the same ratio, it showed an intermediate level between amino acids and carnosine, and it was judged that its protein binding inhibitory power was about 50% lower than that of carnosine. LCGF has a 50 mM undiluted solution, which cannot be argued in the same line as 100 mM of carnosine, but at the same mixing ratio (Polyphenon 60 0.05%), strong curtain formation is still observed, so it has a carnosine blocking activity. It was judged that there is a high possibility that it will surpass.

(Example 4: Examination of protein binding block by carnosine of EGCG)
The above results are the results for Polyphenon 60, which contains a high concentration of catechin, but here we examined it with EGCG, which is a pure catechin product. EGCG was prepared as a physiological saline solution adjusted to 1% of undiluted solution = 0.00218 mol / ml, and three-step concentrations of x10-fold and x100-fold dilution were prepared and used as an index of protein binding of EGCG. Carnosine was adjusted to 0.1 M = 100 mM, mixed with EGCG as shown in Table, and reacted at room temperature for 3 hours. MC was added to the reaction complex, and the appearance precipitate was scored, and the ratio of EGCG to Carnosine in which the protein block was completely formed was calculated based on the judgment.

(Note): EGCG had strong protein binding. That is,
EGCG 20 μl (0.436 μmol): (Carnosine) 0.18 ml = 0.018 m mol = 18 μmol: Judgment; Effective
EGCG 20 μl (0.436 μmol): (Carnosine) 0.018 ml = 0.0018 m mol = 1.8 μmol: Judgment; Not effective

41.3 molecules of Carnosine are required to block the protein binding of one molecule of EGCG. (One Carnosine molecule can accommodate about 0.0243 molecules of EGCG).

(Example 5-1: Cytopathic effect of a complex whose protein binding is blocked by carnosine)
As the cultured cells, mouse Hybridoma (ATCC HB-9848) cells used for producing a monoclonal antibody were used. These cells were seeded in 24 cluster wells at 1.0 x 10 ⁵ / 0.5 ml / well, and 0.05 ml of a separately prepared catechin-carnosine complex ((10: 1) was added to the culture system and cultured at 35 ^o C. , Aged cell degeneration was observed and the degree of cell degeneration was scored in 5 stages from 0 (unmodified) to 4 (100% denatured). Complex was created by carnosine low dose (0.1% Polyphenon 60 0.1 ml +). Two types of 1 M carnosine 0.01 ml + saline 0.09 ml) and high dose (0.1% Polyphenon 60 0.1 ml + 1 M carnosine 0.09 ml + saline 0.01 ml) were prepared and diluted as shown in Table. Polyphenon 60 The concentration was shown as the final concentration after addition to the culture system.

(Note): Polyphenon 60 had a final concentration of 0.00004545%, and the cytopathic effect almost disappeared. From the results so far, the complex prepared with high concentration of carnosine retained the cytopathic effect almost the same as that of Polyphenon 60, although the protein binding activity was 100% lost.

(Example 5-2: Cytopathic effect of carnosine complex)
The same experiment was repeated and reconfirmed.

(Note): From the results of this experiment, it was found that the cell degenerative effect of Polyphenon 60 was very strong, and the final concentration was between 0.00004545% and 0.000004545%. The complex using high-concentration carnosine also retained the same degree of metamorphism as Polyphenon 60 alone. The culture system supplemented with Carnosine alone showed no cell degeneration effect.

(Example 5-3: Examination of concentration of cytopathic effect)
Based on the above results, the more detailed concentration was reexamined below based on Polyphenon 60 with a final concentration of 0.00004545%.

(Note): The carnosine-catechin complex maintained a stronger cell degenerative effect than Polyphenon alone in 5 to 7 days. In particular, even in a test culture system in which Complex was diluted x40 times (Polyphenon 60 corresponds to a low concentration of 0.00001136%), a moderate cell degeneration effect remained.

(Example 5-4: Cytopathic activity of EGCG and carnosine complex)
So far, we have seen the cell metamorphism of Polyphenon 60, which is not a pure catechin product, but we investigated whether the same effect can be seen with EGCG, which is a pure catechin product. For comparison, Polyphenon 60 was prepared using the complex treated as before as a reference. Mix 0.018 ml of 1 M-carnosine with 20 μl of 1% EGCG or Polyphenon 60, react at room temperature for 3 hours, and add 0.162 ml of physiological saline (control is 0.18 ml) × 10 times Complex sample ( And control), the following 2-fold diluted complex was added to the test cell culture well in an amount of 50 μl each, and monitored on a daily basis.

(Note): The results of this experiment provided extremely important information. Comparison of Polyphenon 60 and complex shows that the formation of a complex with carnosine has a stronger cell denaturing effect than catechin alone (in the experimental well of sample x 40-fold dilution, control changed to denaturation level 0 on day 8. Despite this, complex still maintained 4 levels) was shown as a stronger effect when using EGCG. The EGCG / carnosine complex was still maintained at the strongest level of 4 until day 5 in the experimental well diluted sample x 80 times, after which the EGCG alone control was still 2-1 despite the complete disappearance of denaturation. Clear degeneration remained.

The present invention can be used in the food industry, the pharmaceutical industry, and the like.

A string-like B cloth-like C string-like mass D mass

Claims (5)

Ca Runoshin, glutathione and / or as an active ingredient a hepatocyte growth factor, a biologically active maintenance of catechins and non-specific binding inhibiting composition of the protein to the catechins. Ca Runoshin method glutathione and / or with a hepatocyte growth factor, inhibit the non-specific binding of proteins to catechins, and to maintain the biological activity of catechins. A method for measuring the degree of protein binding to catechins based on the shape of a precipitate of methyl cellulose and catechins. A method for searching for an amino acid or a peptide consisting of 2 to 3 amino acids that blocks an unspecified number of protein bonds to catechins by the method of claim 3 . A method of maintaining the cytopathic effect of cultured cells in a state in which the protein binding property of an unspecified number of catechins is lost by forming a complex of catechins and carnosine.