JP2017178796A - Anti-inflammatory additive - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a food additive composition that inhibits inflammation, involved in many diseases, from raw materials widely available as food ingredients.SOLUTION: A nitrogen compound selected from an amino acid, a peptide or a protein is modified with phytic acid, which can obtain a food additive composition having a strong anti-inflammatory activity. The food additive composition in the present invention can be used widely in the form of added to food or feed.SELECTED DRAWING: Figure 1

Description

  The present invention relates to an anti-inflammatory additive for imparting an anti-inflammatory effect to foods, feeds, pharmaceuticals and the like.

“Inflammation” is an essential defense reaction for the living body caused by the immune system against stimuli that are undesirable for living tissues such as foreign body invasion and tissue damage. However, when an immune system's self-reaction or excessive reaction occurs, it becomes a harmful disease that causes damage and pain to the living body itself.
Inflammation is known to be involved in numerous diseases such as cancer, lifestyle-related diseases, autoimmune diseases, neurological diseases, obesity, inflammatory bowel disease, osteoarthritis, rheumatism, and sarcopenia. It has been demanded.

  Inflammation is a state in which immune-competent cells and T cells of the innate immune system accumulate in tissues, causing vasodilation and increased vascular permeability, and causing swelling and pain. In addition, systemic inflammation causes fever, and these reactions occur in the tissue, resulting in tissue dysfunction.

In the early stage of inflammation, neutrophils treat foreign substances such as bacteria, viruses, and dead cells, but in later stages of inflammation, macrophages process by phagocytosis. Macrophages that encounter these foreign substances are activated by antigen presentation and release cytokines such as TNF-α, IL-1β, and IL-6 and chemokines such as IL-8.
Inflammatory cytokines increase vascular permeability and cause fever, redness, and swelling. Chemokines also increase leukocyte chemotaxis and cause swelling. Macrophages play an important role in maintaining homeostasis, but excessive inflammatory responses caused by abnormal macrophage activation can affect many diseases. Therefore, development of a material having an anti-inflammatory action is demanded.

Phytic acid is a kind of biological material and is a hexaphosphate ester of myo-inositol. It is a storage form of phosphorus present in many plant tissues such as seeds, and has a strong chelating action and exists in the form of phytate in which many metal ions are bound in nature. This phytate is called phytin. It has been reported that phytic acid has an antitumor effect (Non-patent Document 1).
In addition, urinary calculi (Non-patent Document 2), prevention of kidney stones (Non-patent Document 3), suppression effect of plaque formation (Non-patent Document 4), and the like have been reported. As for the anti-inflammatory action, inflammatory cytokine inhibitory effect in human colon cells and macrophages (Non-patent Document 5) and edema inhibitory effect in rat carrageenin paw edema test (Non-patent Document 6) have been reported.

J Nutr. 2003 Nov; 133 (11 Suppl 1): 3778S-3784S. Anticancer res., Vol.19 No.5A, pp3717-3722, 1999 Front Biosci. 2007 Jan 1; 12: 2580-7. Caries Res. 1982; 16 (6): 472-4. Dig Dis Sci (2007) 52: 93-102 Indian Journal of Experimental Biology Vol.42, February 2004, pp.179-185

Phytic acid has functionalities such as an antitumor effect, an antioxidant effect, and an anti-inflammatory effect. However, phytic acid, on the other hand, has a strong chelating action, so in the living body, it immediately binds to a cation and forms an insoluble salt with poor reactivity. Therefore, it is considered necessary to ingest a sufficient amount in order to exert the effect.
On the other hand, the strong chelating action of phytic acid itself is highly irritating to the living body, and it has been reported that adverse effects of mineral absorption inhibition and growth inhibition have been reported.

  Then, this invention makes it a subject to provide the technique for exhibiting the physiological function which phytic acid has effectively.

  The present inventor has been diligently examining this problem, and that a conjugate in which phytic acid is directly bound to an amino organic nitrogen compound such as a peptide without using a mineral exhibits a strong anti-inflammatory action in vivo. The inventor found that the effect is superior to phytic acid alone in the living body and completed the present invention.

That is, the present invention includes the following inventions.
(1) An anti-inflammatory additive comprising as an active ingredient a phytic acid conjugate in which an amino group of an amino organic nitrogen compound is bonded to a phosphate group of phytic acid or a salt thereof,
(2) The anti-inflammatory additive according to (1), wherein the amino organic nitrogen compound is selected from amino acids, peptides or proteins, or salts thereof,
(3) The anti-inflammatory additive according to (1) or (2), which is a food additive, a pharmaceutical additive or a feed additive,
(4) The amino group of the amino organic nitrogen compound and the phosphate group of phytic acid or its salt were bound by adjusting the mixture of the amino organic nitrogen compound and phytic acid or its salt to pH 5.5 or lower. A method for producing an anti-inflammatory additive, characterized by obtaining a phytic acid conjugate,
(5) The amino group of the amino organic nitrogen compound and the phosphate group of phytic acid or its salt were bound by adjusting the mixture of the amino organic nitrogen compound and phytic acid or its salt to pH 5.5 or lower. A method for imparting anti-inflammatory activity to the amino organic nitrogen compound, which comprises obtaining a phytic acid conjugate.

  According to the present invention, an anti-inflammatory additive having an excellent anti-inflammatory action based on phytic acid can be provided and used as a food additive, a pharmaceutical additive, a feed additive, or the like.

It is a graph which shows the anti-inflammatory effect with respect to the mouse | mouth macrophage cell of the protein-phytic acid conjugate | bonded_body obtained in Example 1 (Example 5). It is the graph which showed the body weight change of each group during the test period in the animal test (Example 6) of an intestinal inflammation model. It is the graph which showed the expression level of the inflammatory cytokine (TNF- (alpha)) in each group in the animal test (Example 6) of an intestinal tract inflammation model. It is the graph which showed the expression level of the inflammatory cytokine (IL-1 (beta)) in each group in the animal test (Example 6) of an intestinal inflammation model. It is the graph which showed the expression level of the inflammatory cytokine (IL-6) in each group in the animal test (Example 6) of an intestinal inflammation model.

  The anti-inflammatory additive of the present invention is characterized in that a phytic acid conjugate in which an amino group of an amino organic nitrogen compound is bonded to a phosphate group of phytic acid or a salt thereof is an active ingredient. Hereinafter, embodiments of the present invention will be specifically described.

(Amino organic nitrogen compounds)
In the present invention, the amino organic nitrogen compound refers to an organic compound in which nitrogen is contained in the molecule in the state of an amino group (—NH ₂ ). Examples of amino organic nitrogen compounds include proteins (polypeptides), low molecular peptides, amino acids and the like. As the amino organic nitrogen compound, a natural product containing the amino organic nitrogen compound can be used, or a concentrated or purified product of a natural product, or a synthetic compound can be used.

  Protein refers to a high molecular compound obtained by polymerizing amino acids in a chain regardless of animality, plantiness, or synthesis, and is sometimes referred to as a polypeptide. For example, animal proteins include milk-derived proteins (casein, whey protein, etc.), egg-derived proteins such as egg yolk and egg white, meat-derived proteins such as beef, pork, chicken, and fish (collagen, gelatin, etc.), yeast, bacteria, Examples include microorganism-derived proteins such as filamentous fungi. Examples of vegetable proteins include beans-derived proteins such as soybeans and peas, grains-derived proteins such as wheat, barley, rice and rice bran, and seed-derived proteins such as canola and nuts, but are not limited to these examples. It is not a thing.

  Peptide refers to a product obtained by degrading the above protein by an enzyme or chemical treatment to lower the molecular weight than the protein, or a product obtained by artificially synthesizing a plurality of amino acid molecules.

  Examples of amino acids include 20 kinds of α-amino acids (amino acids having an amino group bonded to carbon to which a carboxyl group is bonded) constituting a protein in a living body. Among α-amino acids, there are D-type and L-type optical isomers, any of which may be used. β-amino acids such as β-alanine, γ-amino acids such as γ-aminobutyric acid, ornithine, citrulline, sarcosine, creatine, opine, trimethylglycine, theanine, tricolominic acid, kainic acid, cystine, hydroxyproline, hiroxylysine, thyroxine Amino acids other than α-amino acids such as O-phosphoserine and desmosine can also be used.

(Phytic acid or its salt)
Phytic acid is a hexaphosphate ester of myo-inositol. Examples of phytic salts include alkali metal salts and alkaline earth metal salts of phytic acid such as sodium phytate and calcium phytate, but are not limited to these examples. In the present invention, any form of phytic acid and phytate can be used, and it can be appropriately selected in consideration of handling properties and the production process of the composition.
The phytic acid used in the present invention may be naturally derived, for example, extracted from plants such as rice, soybeans and wheat. Moreover, what was synthesize | combined by the chemical synthesis method etc. and a reagent can also be used.

(Phytic acid conjugate)
The phytic acid conjugate in the present invention is a combination of the above amino organic nitrogen compound and phytic acid or a salt thereof, but is not an indirect chelate bond via a cation such as calcium, but an amino organic organic compound. This refers to a direct combination of an amino group of a nitrogen compound and a phosphate group of phytic acid (hereinafter sometimes referred to as “the present conjugate”). In the case of a phytic acid conjugate via a chelate bond by a cation, it is foreseen that when this is taken orally, the bond is easily released by gastric acid. This is because the chelate bond is easily detached under acidic conditions.
On the other hand, since this conjugate is a direct linkage, it is relatively stable in vivo. Such a direct bond does not occur simply by mixing an amino organic nitrogen compound and phytic acid, and is limited to a part derived from plants represented by seeds in nature.

  More simply, this conjugate can be obtained through an artificial modification step in the presence of an amino organic nitrogen compound and phytic acid.

One embodiment of a method for obtaining the present conjugate through an artificial modification step can be performed as follows.
First, for example, an amino organic nitrogen compound and phytic acid or a salt thereof are mixed in an aqueous system to obtain a mixture, and both compounds are allowed to coexist. Next, the pH of the mixture is adjusted to an acidity of 5.5 or less. The upper limit of the pH is further preferably 5 or less, 4.5 or less, 4 or less, 3.5 or less, or 3 or less. The lower limit of the pH is not particularly limited, but can be 1 or more. After pH adjustment, the pH is maintained with stirring as necessary. Although the maintenance time is not particularly limited, it may be maintained for a time sufficient for the production of the present conjugate. For example, the maintenance time is 10 minutes to 24 hours, preferably 10 minutes to 3 hours. Next, with this pH, or if necessary, raise the pH to the alkaline side with an alkali and neutralize it to, for example, about pH 6-8, and then centrifuge the mixture to collect the precipitate. A fraction containing the body is obtained.

  However, since the amino organic nitrogen compound is a low molecule, the precipitate can be concentrated by fractionation means such as membrane fractionation if precipitation does not occur even when the pH is adjusted to acidic. Alternatively, after obtaining a conjugate with phytic acid in the state of a high molecular protein, it may be decomposed into a low molecular weight peptide or amino acid by an enzyme such as a protease.

  The content of phytic acid in the conjugate is not particularly limited because the saturation amount of phytic acid that can be directly bonded varies depending on the composition of the amino organic nitrogen compound.

  By directly bonding the amino group of the amino organic nitrogen compound and the phosphate group of phytic acid by the method as described above, anti-inflammatory ability can be imparted to the amino organic nitrogen compound.

(Anti-inflammatory additive)
The anti-inflammatory additive of the present invention comprises the above-mentioned conjugate as an active ingredient, and the conjugate itself can be used as an anti-inflammatory additive, and if necessary, other raw materials such as excipients Can also be mixed. Also, other active ingredients having an anti-inflammatory action can be combined.

  The anti-inflammatory additive of the present invention can also be used in various fields such as food additives, pharmaceutical additives, feed additives, external skin additives, cosmetic additives and the like. Further, the subject to which the anti-inflammatory additive is used is not particularly limited. For example, in addition to humans, primates (chimpanzees, monkeys, etc.), livestock animals (cattle, pigs, chickens, horses, etc.), pet animals ( It can be used as an additive for ingesting active ingredients to mammals such as dogs and cats) and laboratory animals (such as mice, rats and rabbits), birds and other animals.

  The anti-inflammatory additive of the present invention can effectively prevent or treat inflammatory diseases or symptoms. Since the additive reduces or suppresses the production of inflammatory cytokines in vivo, it is particularly effective for inflammatory diseases or symptoms caused by inflammatory cytokines. Such symptoms include inflammatory bowel disease, obesity, rheumatoid arthritis, osteoarthritis, sarcopenia, chronic inflammation, arteriosclerosis, lifestyle-related diseases and inflammation due to obesity.

  Hereinafter, embodiments of the present invention will be described more specifically with reference to examples. The unit “%” or “part” means “% by weight” or “part by weight” unless otherwise specified.

(Example 1) Preparation of protein-phytic acid conjugate As described below, phytic acid modification was performed on a protein to prepare a protein-phytic acid conjugate.
Two aqueous solutions of 6.3 L sodium caseinate and separated soy protein were prepared so as to obtain a 0.792% aqueous solution concentration, and the pH was adjusted to 2 with hydrochloric acid. 6.3 L of 400 μM sodium phytate aqueous solution adjusted to pH 7 with sodium hydroxide was mixed. Immediately readjusted to pH 2 with hydrochloric acid and stirred at room temperature for 30 minutes. After centrifugation at 6,000g for 15 minutes, the precipitate was collected. The precipitate was washed with 10-fold diluted phosphate buffered saline (PBS (-) solution), and then centrifuged again at 6,000 g for 15 minutes to collect the precipitate. The precipitate was lyophilized to obtain Modified Form 1 and Modified Form 2 in which phytic acid was bound to sodium caseinate and separated soybean protein, respectively.
In the acidic region, phytic acid and cation are not bonded, and the phosphate group of phytic acid is released. On the other hand, in the acidic region below the isoelectric point of the protein, the amino group of the protein is charged with a positive charge, and it is considered that the protein and phytic acid conjugates were formed as a direct bond and formed as a precipitate.

(Example 2) Preparation of Artificial Digestion Artificial digestion products of sodium caseinate and separated soybean protein, modified body 1 and modified body 2 obtained in Example 1 were prepared as follows.
Four aqueous solutions of sodium caseinate, separated soybean protein, modified body 1 and modified body 2 were prepared so as to have a 2.5% aqueous solution concentration, and the pH was adjusted to 2 with hydrochloric acid. Pepsin was added to 1% of the substrate and reacted at 37 ° C. for 2 hours.
After adjusting to pH 8 with sodium hydroxide, trypsin was added to the substrate 1.7% and α-chymotrypsin was added to the substrate 0.03% and reacted at 37 ° C. for 2 hours. After inactivating the enzyme by boiling for 5 minutes, centrifugation was performed at 1,000 g for 10 minutes, and the supernatant was recovered and freeze-dried to obtain 4 types of artificial digests.

(Example 3) Measurement of phytic phosphorus content The phytic phosphorus content of the four types of artificial digests obtained in Example 2 was measured as follows.
8 ml of an aqueous solution was prepared so that the protein content was about 2%. 2 ml of 15% trichloroacetic acid aqueous solution was added to the aqueous solution and stirred for 15 minutes. The aqueous solution was centrifuged at 3,000 g for 15 minutes, and then the supernatant was filtered through a 0.45 μm filter.
To 5 ml of the supernatant, ₃ ml of 1% FeCl ₃ .6H ₂ O and 5% Na ₂ SO ₄ (in 1 M hydrochloric acid) solution were added and boiled for 30 minutes. Next, the solution was centrifuged to remove the supernatant, and 5 ml of 3% trichloroacetic acid aqueous solution was added to the precipitate and stirred.
The solution was centrifuged to remove the supernatant, and then 5 ml of 0.17M hydrochloric acid was added to the precipitate and stirred. The solution was centrifuged to remove the supernatant, and 5 ml of warm ultrapure water was added to the precipitate and stirred.
The solution was centrifuged to remove the supernatant, and 0.5 ml of 60% perchloric acid was added to the precipitate to suspend it. The solution was dispensed into a weighed test tube and heated at 180 ° C. for 2 hours.
Next, 2 ml of 2M hydrochloric acid was added, followed by boiling for 30 minutes. After adding phenolphthalein to the solution, it was neutralized with 20% sodium hydroxide and the weight of the test tube was measured.
400 μl of ultrapure water was added to 100 μl of the sample solution and the standard solution for calibration curve (KH ₂ PO ₄ , 0 to 200 μg / ml) and stirred. 50 μl of 60% perchloric acid was added and stirred.
150 μl of an ammonium vanadate solution (dissolved in 1.17 g of ammonium metavanadate in warm ultrapure water, 20 ml of 60% perchloric acid after cooling, and made up to 500 ml with MiliQ) was added to the solution and stirred. 300 μl of a 3.53% ammonium molybdate solution (35.3 g of ammonium molybdate dissolved in ultrapure water and made up to 1 L) was added to the solution and stirred.
The solution was allowed to stand at room temperature for 30 minutes, and then 200 μl was dispensed into a 96-well plate, and the absorbance at 420 nm was measured. The content of phytic acid (MW = 660.8) was calculated from the total phosphorus amount calculated from the phytic acid measurement solution. The measurement results are shown in Table 1.

(Table 1) Phytic phosphorus content

(Discussion)
As shown in Table 1, the phytic acid content after artificial digestion is higher than that of the unmodified protein in both the modified body 1 using sodium caseinate modified with phytic acid binding and the modified body 2 using separated soy protein as a raw material. Were also found to be high. In other words, it was shown that these modified products are bound to protein and phytic acid, and that the binding between protein and phytic acid is maintained even after artificial digestion.

(Example 4) Cell culture for anti-inflammatory evaluation In order to evaluate the anti-inflammatory action of the artificial digest obtained in Example 2 on macrophages, cell culture was performed by the following method.
“J774.1 cells” (provided by RIKEN) were used as mouse macrophage cells. Add bovine serum to RPMI-1640 medium (Wako Pure Chemical Industries, Ltd.) to a final concentration of 10%, and add 100 IU / ml penicillin and 100 μg / ml streptomycin as antibiotics. A culture medium was used.
J774.1 cells were cultured in a cell culture medium at 37 ° C. under 5% CO ₂ , and cultured in a 10 cm petri dish until confluent. Cells that reached confluence were collected, diluted to 2.5 × 10 ⁵ cells / ml, and 1 ml each was added to a 24-well cell culture plate.
The cells were cultured for 24 hours under conditions of 37 ° C. and 5% CO ₂ , and the medium was removed with an aspirator. After removal, 350 μl of artificial digest prepared to a final concentration of 5 μg / ml and 350 μl of “Pam3CSK4” (InvivoGen) as an inflammation inducer to a final concentration of 10 ng / ml were added, The cells were cultured for 24 hours under 5% CO ₂ conditions. Thereafter, 200 μl of the culture supernatant was collected.

(Example 5) Measurement of IL-6 concentration by ELISA As described below, the inflammatory cytokine IL-6 concentration in each medium cultured in Example 4 was measured by ELISA.
“Purified rat anti-mouse IL-6” (primary antibody), “Biotinylated rat anti-mouse IL-6” (secondary antibody), “Recombinant mouse IL-6” (standard IL-6) and “Streptavidin-Alkalin” “Phosphate” was purchased from BD Biosciences and used.
A primary antibody prepared with 0.1 M Na ₂ HPO ₄ buffer (pH 9.0) to a concentration of 0.5 μg / ml was added to a 96-well microplate at 50 μl / well and allowed to stand at room temperature for 2 hours. Next, the plate was washed 3 times with PBS-Tween20 (PBS-T) and thoroughly drained.
Thereafter, 3% BSA in PBS-T was added at 100 μl / well, allowed to stand at room temperature for 1 hour, washed 3 times with PBS-T, and thoroughly drained. A culture supernatant sample diluted appropriately with PBS-T was added in an amount of 50 μl / well, allowed to stand at 4 ° C. for 24 hours, washed 3 times with PBS-T, and sufficiently drained.
Add 50 μl / well of the secondary antibody prepared at 0.5 μg / ml with PBS-T, shield from light with aluminum foil, let stand at room temperature for 1 hour, wash with PBS-T three times, and drain thoroughly. It was. “Streptavidin-Alkalin Phosphate in PBS-T” was added in an amount of 50 μl / well, light-shielded with aluminum foil, allowed to stand at room temperature for 1 hour, washed 3 times with PBS-T, and thoroughly drained.
Put 0.2 mg / ml of disodium 4-ditrophenyl phosphate in 1M diethanolamine buffer at 50 μl / well, light-shield with aluminum foil and let stand for 20 minutes, then 405 nm (reference wavelength 492 nm) with a microplate reader. Absorbance was measured twice and the average value was calculated. The measurement results are shown in FIG.

(Discussion)
As a result of verifying the anti-inflammatory action of each artificial digest, as shown in FIG. 1, the casein sodium artificial digest showed no change compared with the positive control IL-6 production, and the anti-inflammatory action was recognized. I couldn't.
On the other hand, the modified product 1 artificial digest showed a significant decrease in IL-6 production, and an anti-inflammatory effect could be obtained.
In addition, IL-6 production was suppressed in the isolated soybean protein artificial digest even before phytic acid modification, but IL-6 production was significantly reduced in the modified 2 artificial digest, which modified phytic acid. It was shown that the anti-inflammatory action was enhanced.

(Example 6) Intestinal inflammation animal test In order to evaluate the anti-inflammatory action in vivo, an animal test of a mouse intestinal inflammation model using dextran sulfate sodium (DSS) was performed.
Mice (C57BL / 6, female, 7 weeks old) were purchased from Japan SLC, and the acclimatized feed was “CRF-1” (produced by Oriental Yeast Co., Ltd.). Gave. Drinking water was given ion exchange water.
Mice were allowed to freely feed and drink and were kept under a 12-hour light period. All animals were preliminarily raised for 3 days before being used in this experiment, and the untreated group (n = 6), DSS administered group (n = 6), modified group 1 (n = 6), phytic acid group (n = Divided into 4 groups of 6).
For 5 days after acclimatization, the non-administered group and the DSS-administered group had ion exchange water, the modified group 1 had a 0.5% aqueous solution of the modified substance 1 artificial digest described in Example 2, and the phytic acid group had 0.0285% phytic acid. An aqueous sodium solution was allowed to drink freely.
Thereafter, 2.25% of DSS (purchased from MP Biochemicals, Mw 36,000-50,000) was added to the DSS-administered group, modified group 1 and phytic acid group free drinking water solution to allow free drinking. FIG. 2 shows the change in body weight of each group during the 15-day test period.
Dissection was performed 11 days after the start of DSS administration. In dissection, colon tissue was collected, RNA was extracted, and the expression levels of inflammatory cytokines (TNF-α, IL-1β, IL-6) were measured. The measurement results of each cytokine are shown in FIGS.

(Discussion)
As shown in FIG. 2, intestinal inflammation was caused by DSS free drinking, and body weight was significantly reduced in the DSS administration group. On the other hand, the phytic acid group showed a slight effect of suppressing weight loss. However, in the modified group 1 in which the same amount of phytic acid as that of the phytic acid group was modified, a stronger weight loss suppression effect was confirmed than in the phytic acid group.
In addition, as shown in FIGS. 3 to 5, in the expression of inflammatory cytokines, TNF-α and IL-6 had no effect in the phytic acid group, whereas in the modified body 1 group, the phytic acid group in all cytokines The expression suppression effect was stronger than that. That is, in the living body, phytic acid alone has a low anti-inflammatory effect, but even if the amount is the same, a more prominent anti-inflammatory effect was shown by using a conjugate of protein and phytic acid.

Claims (5)

An anti-inflammatory additive comprising, as an active ingredient, a phytic acid conjugate in which an amino group of an amino organic nitrogen compound is bound to a phosphate group of phytic acid or a salt thereof. The anti-inflammatory additive according to claim 1, wherein the amino organic nitrogen compound is selected from amino acids, peptides or proteins, or salts thereof. The anti-inflammatory additive according to claim 1 or 2, which is a food additive, a pharmaceutical additive or a feed additive. A phytic acid bond in which an amino group of an amino organic nitrogen compound and a phosphate group of phytic acid or a salt thereof are bonded by adjusting a mixture of the amino organic nitrogen compound and phytic acid or a salt thereof to pH 5.5 or lower. A method for producing an anti-inflammatory additive, characterized by obtaining a body. A phytic acid bond in which an amino group of an amino organic nitrogen compound and a phosphate group of phytic acid or a salt thereof are bonded by adjusting a mixture of the amino organic nitrogen compound and phytic acid or a salt thereof to pH 5.5 or lower. A method for imparting anti-inflammatory ability to the amino organic nitrogen compound, which comprises obtaining a body.

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