KR20160059088A - A functional composition for improving inflammation consisting of branched chain amino acids as an efficient component - Google Patents

Abstract

Provided in the present invention are functional food, animal feed and a pharmaceutical composition comprising branched amino acid as an active component for anti-inflammation based on verification by measuring cytotoxicity and measuring an NO generation inhibiting effect and an inflammation controlling effect against cytokine.

Description

[0001] The present invention relates to an anti-inflammatory functional composition comprising branched amino acid as an active ingredient,

The present invention relates to an inflammation-improving functional composition comprising a branched amino acid as an active ingredient. More particularly, the present invention relates to a composition for improving inflammation, which comprises measuring cell survival rate by MTT assay, evaluating the inhibitory effect on NO production, Improved functional food, animal feed, and pharmaceutical composition.

Inflammatory disorders are one of the most important health problems in the world. Inflammation is one of the defense mechanisms of biological tissue caused by host invasion, such as foreign substances or stimuli. Its causes are infectious causes such as bacteria, viruses and parasites, and physical causes such as burns or radiation. In addition, chemical agents such as toxins, drugs and industrial agents, and immune responses such as allergies and autoimmune reactions are also associated with oxidative stress. When inflammatory reactions occur, various inflammatory mediators are formed, which can cause clinical symptoms such as fever, redness, pain, swelling and dysfunction. Harmful stimuli such as inflammatory factors such as LPS (lipopolysaccharide) overexpress the human immune system causing prostaglandin E2 (prostaglandin E ₂ , PGE ₂ ), IL-1 (interleukin-1), IL-6 6) and nitric oxide (NO), which are known to induce various inflammatory diseases and immune diseases (Lee et al., Methanol extracts of Stewartia koreana inhibited cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) gene expression by blocking NF-kappa B transactivation in LPS-activated RAW 264.7 cells, Mol. Cells 30: 398-404, 2007; Nishida et al., Geranylacetone induces cyclooxygenase-2 expression in cultured rat gastric epithelial cells through NF-κB, Dig. Dis. Sci. 52: 1890-1896, 2007).

In particular, NO is produced in endothelial cells or macrophages in normal state, and it has various physiological activities besides cell killing and sterilization. It is known that NO acts to maintain homeostasis of blood pressure in relaxation of vascular endothelial cells. The stimulation of LPS, inflammation inducers and irradiation induces the induction of inducible nitric oxide synthetase (iNOS), which is an inducible nitric oxide synthase, to produce a large amount of NO continuously. Leading to a variety of inflammatory and immune diseases. That is, the production of NO by the expression of iNOS functions as a typical inflammatory factor of immune cells (Guslandi, Nitric Oxide and inflammatory bowel diseases, Eur. J. Clin. Invest. 28: 904-907, 1998). Therefore, the development of iNOS activity inhibitor as a therapeutic agent for inflammatory diseases is highly likely to be developed.

 Other inflammatory factors are inflammatory cytokines such as tumor necrosis factor-α, TNF-α, interleukin-1β, interleukin-6, IL- 6), monocyte chemoattractant protein-1 (MCP-1), and the like. TNF-α, which is found as a tumor necrosis factor, is associated with autoimmune diseases such as rheumatoid arthritis, rejection of organ transplantation, allergic inflammatory diseases such as asthma and atopic dermatitis, acute immune diseases such as sepsis and acute liver disease (MCP-1 expression by suppressing the expression of p38 MAPK and NF-κB pathways in rat vasculature), and inhibiting the synthesis of TNF-α (Zhong et al. Analysis of GEO datasets, Joint Bone Spine 81: 325-330 (2001), pp. 167-160, 2012. Kim et al., Gene expression profile predicting response to anti-TNF treatment inhibitors with rheumatoid arthritis , 2014).

However, existing anti-inflammatory drugs that can treat inflammatory diseases caused by these various mechanisms are classified into steroidal anti-inflammatory agents and non-steroidal anti-inflammatory agents. These anti-inflammatory agents are synthetic agents with adverse effects in the human body. Is required.

The branched-chain amino acids are essential amino acids composed of leucine, valine and isoleucine. They are amino acids that should be ingested as foods because they are not synthesized in the body but are important for the life maintenance of the human body. Molecular amino acids constitute about one-third of the constituents of muscle proteins and are known to be amino acids that account for 40% of the amino acids required by mammals (O'Connell, The complex role of branched chain amino acids in diabetes and cancer, Metabolites 3: 931-945, 2013). Branch amino acids have a geometric structure consisting of branched aliphatic chains (Simone et al., Branched-chain amino acids influence the immune properties of microglial cells and their responsiveness to pro-inflammatory signals. Biochimica, Biophysica. Acta 1832: 650-659, 2013) Unlike other amino acids, various inventions have been carried out as well as having a unique chemical structure. Branch amino acids have been shown to affect gene expression, protein metabolism, apoptosis, and insulin resistance, and have been shown to play an important role in brain function (Tagiri et al., Branched-chain amino acids in liver diseases, World J. Gastroenterol 19: 7620-7629, 2013).

An antioxidant functional food, feed, and pharmaceutical composition containing a branched amino acid as an active ingredient, which has been proposed by the present inventor as a prior art of the present invention, is disclosed in Application No. 10-2014-0123655, but mainly comprises leucine, valine, isoleucine And a branched amino acid composed of at least one branched amino acid. Also disclosed is a pharmaceutical composition containing a branched amino acid and a preparation method thereof, which is disclosed in Korean Patent No. 10-1458670. However, it is also possible to use a branched amino acid as an active ingredient, To pharmaceutical liquid suspension compositions and methods for their preparation. An anti-inflammatory agent including a spine herb extract is disclosed in Korean Patent No. 10-1185903. However, it is not cytotoxic, but also has NO secretion amount, iNOS and COX-2 expression, iNOS and COX-2 mRNA transcription, The present invention relates to an anti-inflammatory agent which inhibits effective inflammation by detecting the transcription of various cytokines related to inflammation.

On the other hand, a composition for improving anti-inflammatory, anti-allergy and anti-wrinkle properties comprising an extract of Zucchini Leaves or 4-O-methyl Honokiol isolated therefrom is disclosed in Korean Patent No. 10-1116852, Inhibition of iNOS activity, inhibition of iNOS and COX2 gene expression, inhibition of PGE2, IL-6 and IL-8 production, enhancement of antihistamine activity, inhibition of elastase activity of 4-O-methylhonokiol An anti-inflammatory, anti-inflammatory, anti-inflammatory, anti-inflammatory, anti-inflammatory, anti-inflammatory and anti-wrinkle effect.

Therefore, no patent invention discloses an anti-inflammatory functional composition containing three branched amino acids such as leucine, valine and isoleucine as active ingredients.

Accordingly, an object of the present invention is to provide an inflammation-improving functional composition containing a branched amino acid as an active ingredient, since the cell survival rate is measured by MTT assay, the inhibitory effect on NO production is evaluated, and the effect on expression of anti- have.

The present invention relates to a method for the treatment and prophylaxis of cancer, comprising: culturing a mouse macrophage Raw 264.7 cell line and measuring cell survival rate through MTT assay; Confirming the cytotoxicity and the inhibition of NO production by confirming the effect of the triplet branched amino acid in the above step on NO production; Cytokine was measured by real-time PCR to confirm the effect of the three branching amino acids on the expression of anti-inflammatory genes.

The present invention has an excellent effect of providing an inflammation-improving functional composition containing a branched amino acid as an active ingredient by measuring the cytotoxicity of a branched amino acid, the inhibitory effect on NO production and the anti-inflammatory effect on cytokine.

FIG. 1 is a graph showing the cell survival rate of the Raw 264.7 cell line, which is a macrophage when treating three kinds of branched amino acids according to the present invention.
FIG. 2 is a graph showing the effect of three kinds of branched amino acids according to the present invention on NO production of Raw 264.7 cell line activated by LPS.
FIG. 3 is a graph showing the expression of COX-2 (A), IL-6 (B), MCP-1 (C), TNF-α (D), and TLR in Raw 264.7 cell line in which three kinds of branched amino acids according to the present invention are activated by LPS (E), TLR-2 (F), IL-10 (G), IL-1β (H), IL-2 The graph shows the effect.
FIG. 4 is a graph showing the effect of three kinds of branched amino acids according to the present invention on the expression of iNOS in LPS-activated Raw 264.7 cell line.

The branched amino acids according to the present invention were purchased from Cremar (Gyeonggi-do, South Korea). MTT (3- [4,5-dimethythiazol-2-yl] -2,5-dipheny l tetrazolium bromide) reagent for measuring cell viability was purchased from Sigma Chemical Co. The product was purchased. Hyclone Laboratories products such as DMEM (Dulbecco's Modified Eagle Medium) medium, FBS (fetal bovine serum) and penicillin-streptomycin were used for cell culture, and LPS (lipopolysaccharides from Escherichia coli O111: B4), sodium nitrite, sulfanilamide and N- (1-naphthyl) ethylenediamine dihydrochloride were purchased from Sigma. All reagents used in other experiments were of grade 1 or higher.

In the present invention, the above-mentioned three branched amino acids are used singly for the purpose of verifying the physiological activity, but they may be used by mixing one or more of them.

Hereinafter, the present invention will be described in detail with reference to examples. The following examples are for illustrative purposes only and are not intended to limit the invention.

Example  1. Cell line and culture

The Raw 264.7 cell line, which is a macrophage cell line of the mouse according to the present invention, was used from Korean Cell Line Bank (KCLB).

For the cell culture of the present invention, 10% fetal bovine serum (FBS), 100 units / mL penicillin and 100 μg / mL streptomycin were added to DMEM (Dulbecco's Modified Eagle Medium) medium and 95% And cultured in a 5% CO 2 incubator (MCO-18AIC, SANYO) at 37 ° C.

Example  2. MTT assay Cell survival rate

MTT assay was performed to measure the cytotoxicity of Raw 264.7 cell line of three kinds of branched amino acids according to the present invention. The activated cell line was adjusted to 2 × 10 ⁵ cells / mL and 100 μL each was added to 96-well plates. The cells were preincubated for 24 hours at 37 ° C. in a 5% CO 2 incubator. The final concentration was 1 μg / mL of LPS and 12.5 mM , 25 mM, 50 mM and 100 mM of the branched amino acids valine, isoleucine and leucine, respectively. After further incubation for 44 hours, 50 μL of 2.5 mg / mL MTT solution dissolved in PBS buffer was added to each well and further incubated for 4 hours to induce formazan formation. After the reaction was completed, the supernatant was removed and 200 μL of DMSO was added to dissolve the formazan formed on the well bottom. Absorbance was measured at 570 nm using an ELISA reader (Model 680, BioRad, USA).

Calculating Equation 1 yielded% cell viability.

Experimental Example  1. Cytotoxicity measurement of three amino acid bases

MTT assay was performed to confirm the cytotoxicity of Raw 264.7 cell line of three branched amino acid mouse macrophages according to the present invention. Experimental results showed that cell viability exceeding 98% was observed in all of the three kinds of branched amino acids at concentrations of 12.5 mM, 25 mM, 50 mM, and 100 mM, indicating no cytotoxicity against macrophages (FIG. 1).

Example  3. NO  Identify the impact on production

The effects of branching amino acids on NO production in Raw 264.7 cell line, a macrophage activated by LPS, were measured. The cells were preincubated in a 24-well plate at a concentration of 2 × 10 ⁵ cells / well and preincubated for 2 hours. After 2 hours, the cells were treated with basal amino acids 12.5 mM, 25 mM, 50 mM and 100 mM and 1 μg / And then incubated for additional 24 hours. The griess reagent, prepared by adding 100 μL of the same amount of 1% sulfanilamide and 0.1% N- (1-naphthyl) ethylenediamine dihydrochloride in 5% phosphoric acid, After incubation at room temperature, the absorbance was measured at 540 nm using an ELISA reader (Model 680, BioRad). NO concentration in the culture medium was determined using a standard curve for the concentration of sodium nitrite (NaNO ₂ ).

Experimental Example  2. Three species of branched amino acids NO  Measurement of production inhibition effect

The Raw 264.7 cell line, a macrophage, is a cell that participates in the inflammatory response and produces nitric oxide (NO). Physiologically, NO plays a variety of roles such as removing bacteria and tumors, regulating blood pressure, or mediating neurotransmission. However, when the inflammatory reaction occurs, the expression of iNOS is increased in related cells to produce a large amount of NO. Excessively produced NO causes tissue damage, gene mutation and nerve damage, and increases vascular permeability, Promoting the reaction. Therefore, the anti-inflammatory effect can be measured by measuring the degree of inhibition of the production of NO.

The effect of three kinds of branched amino acids on NO production of macrophages is shown in Fig. In the negative control group without LPS treatment, NO production was measured to be 2.28 μM, indicating that LPS did not induce inflammation. In the group treated with LPS at a concentration of 1 μg / mL, NO production was increased to 45.63 μM, and inflammation was induced. In the case of treatment with three kinds of branched amino acids, NO production was decreased. In the group treated with 12.5 mM of leucine, the amount of NO decreased by 15 μM was 29.97 μM. When treated with 25 mM, 50 mM, and 100 mM, the amount of NO was further reduced to 24.47 μM, 15.97 μM, 8.60 [mu] M inhibited NO production. The inhibition rate was 81.14% at 100 mM of leucine. The inhibition rate of valine and isoleucine was lower than that of leucine, but the inhibition rate was also increased with increasing concentration. The inhibition rates of valine and isoleucine at the highest concentration of 100 mM were 29.65% and 42.94%, respectively. .

Example  4. Real - time PCR Lt; RTI ID = 0.0 > ( cytokine ) Measure

IL-2, IL-6, MCP-1, TNF-α, TLR-4, TLR-2, IL-10, IL-1β and IL-2 which are known as inflammation related genes to analyze the anti- , IL-4, IFN-γ, and iNOS were measured by real-time PCR.

The Raw 264.7 cell line was cultured in a 24-well plate at a concentration of 2 × 10 ⁵ cells / well for 24 hours. The first step was to treat each branched amino acid at 100 mM After culturing for 24 hours, the cells were treated with 1 μg / mL LPS for 24 hours. The second condition was that the cells were treated with 100 mM of each branched amino acid and 1 μg / mL of LPS for 24 hours.

The medium was removed from the Raw 264.7 cell line after the reaction and homogenized by adding Trizol reagent. Chloroform was added and centrifuged at 10,000 × g for 15 min. The supernatant was used for RNA isolation. RNA was extracted by adding RNase-free water and isopropanol to the supernatant and cDNA was synthesized using M-MLV reverse transcriptase.

The synthesized cDNA was mixed with 2 × SYBR Green master mix and primer and amplified using a real-time DNA thermal cycler (iQTM5 multicolor real-time PCR detection system). The purity of the amplified product was verified by determining the melting curve of the PCR reaction using the Delta delta Ct method. The primers used for real-time PCR were shown in Table 1 and β-actin was used for the housekeeping gene.

Primer sequence used for real-time PCR analysis Gene Description Primer sequence COX-2 cyclooxygenase-2 forward: 5-tgggaagctttctccaacct-3
reverse: 5-gtgaagtgctgggcaaagaa-3 IL-6 interleukin-6 forward: 5-ccttcctaccccaatttcca-3
reverse: 5-cgcactaggtttgccgagta-3 MCP-1 monocyte chemoattractant protein-1 forward: 5-catctgccctaaggtcttc-3
reverse: 5-cttgaggtggttgtggaa-3 TNF-a tumor necrosis factor-alpha forward: 5-atgagaagttcccaaatggc-3
reverse: 5-gaattttgagaagatgatctgagtg-3 TLR-4 toll-like receptor-4 forward: 5-atagcatggaccttaccggg-3
reverse: 5-ggcttgtattcaaaaggccc-3 TLR-2 toll-like receptor-2 forward: 5-tgatcttgctcgtaggtgcc-3
reverse: 5-ttaaagggcgggtcagagtt-3 IL-10 interleukin-10 forward: 5-taaggctggccacacttgag-3
reverse: 5-agttttcagggatgaagcgg-3 IL-1? interleukin-1 forward: 5-gttgacggacccaaccgat-3
reverse: 5-aaggtccacgggaaagacac-3 IL-2 interleukin-2 forward: 5-acctggagcagctgttgatg-3
reverse: 5-gtcaaatccagaacatgccg-3 IL-4 interleukin-4 forward: 5-atcatcggcattttgaacga-3
reverse: 5-aagcccgaaagagtctctgc-3 IFN-y interferon-γ forward: 5-acactgcatcttggctttgc-3
reverse: 5-ctggctctcaggattttca-3 iNOS inducible nitric oxide synthase forward: 5-cttggtgaggggactggact-3
reverse: 5-ggggttttctccacgttgtt-3 β-Actin forward: 5-gattactgctctggctccta-3
reverse: 5-atcgtactcctgcttgct-3

Experimental Example  3. Measurement of anti-inflammatory gene expression effect of 3 kinds of branched amino acids

The present invention relates to the use of the compounds of the present invention for the treatment and prophylaxis of COX-2, IL-6, MCP-1, TNF-α, TLR-4, TLR-2, IL-10, IL-1β, IL- The results of the effect on the gene expression of IFN-y are shown in FIG. 3 and Table 2. In analyzing the anti-inflammatory gene, the experiment was carried out according to the first condition of Example 4. The expression of COX-2 was significantly increased by LPS treatment and was significantly decreased by treatment with the branched amino acids valine, isoleucine and leucine. In addition, IL-6 gene expression increased by treatment with LPS was reduced by treatment with valine and leucine. MCP-1 did not show any significant change even after LPS treatment, but it showed a significant reduction effect when treated with valine and isoleucine. Expression of TNF-α was increased by treatment with LPS and decreased by valine treatment. Expression of TLR-4 was significantly increased by LPS treatment and decreased by treatment with valine and leucine. Expression of TLR-2 and IL-2 was significantly increased when LPS was treated and significantly decreased when treated with branched amino acid. The expression of other anti-inflammatory genes was also significantly increased in the LPS-treated group, but was significantly decreased in the group treated with the branched amino acid. This suggests that the branched amino acid has a significant effect on the expression of anti-inflammatory genes in the macrophage Raw 264.7 cell line.

Effect of branched amino acid on the expression of anti-inflammatory genes in LPS-activated Raw 264.7 cell line LPS (-) LPS (+) - Valine Isoleucine Leucine COX-2 1.32 ± 1.22 52.96 + - 11.18 3.59 + - 0.41 0.01 ± 0.01 0.19 + - 0.12 IL-6 1.03 + - 0.37 81.15 ± 10.32 21.65 ± 3.38 69.96 ± 30.64 12.14 + - 4.86 MCP-1 1.02 0.30 1.48 0.49 0.42 ± 0.02 0.34 + 0.03 1.37 ± 0.12 TNF-a 1.02 0.30 32.10 + - 4.45 1.12 ± 0.17 52.73 ± 35.99 92.36 ± 32.38 TLR-4 1.00 + - 0.11 5.68 ± 3.42 1.37 + - 0.44 2.02 + 0.44 1.85 ± 0.05 TLR-2 1.00 + 0.06 4.26 ± 0.46 0.21 ± 0.00 0.62 ± 0.56 0.71 + - 0.10 IL-10 1.29 ± 1.14 47.04 ± 20.12 37.51 + - 0.49 9.95 ± 6.20 29.72 ± 7.81 IL-1? 1.07 ± 0.55 259.16 + - 6.86 6.33 ± 1.58 494.56 ± 148.02 301.13 ± 15.66 IL-2 1.00 + 0.06 1.88 + 0.02 0.24 + 0.02 0.28 0.22 0.47 + 0.11 IL-4 1.01 0.20 1.75 + - 0.56 7.66 ± 0.67 1.05 ± 1.10 1.17 + 0.02 IFN-y 1.02 + - 0.28 1.29 + - 0.32 2.31 ± 0.51 0.47 + 0.29 0.42 ± 0.01

In addition, the effect of the branched amino acid on the degree of gene expression of iNOS, an enzyme producing nitric oxide, which is an intracellular inflammatory factor, was tested according to the second condition of Example 4, and the result is shown in FIG. The expression level of iNOS was increased by LPS, which is an inflammation inducer, and the inhibition rate of expression of 34.82% (valine), 17.41% (isoleucine) and 65.96% (leucine) The effect was excellent.

INDUSTRIAL APPLICABILITY As described above, the present invention provides an inflammation-improving functional composition containing a branched amino acid as an active ingredient by measuring the cytotoxicity measurement, the NO production inhibitory effect and the anti-inflammatory effect on the cytokine, In addition to food and animal feed, it is a very useful invention in the pharmaceutical industry for the prevention and treatment of inflammation.

Claims (4)

Pharmaceutical composition for prevention and treatment of inflammation containing branched amino acid as an active ingredient An anti-inflammatory and improving functional food composition containing a branched amino acid as an active ingredient Animal feed composition for prevention and improvement of inflammation containing branched amino acid as an active ingredient The composition according to any one of claims 1 to 3, wherein the branched amino acid is any one or more selected from the group consisting of leucine, valine and isoleucine.

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