KR101483727B1 - Anti-inflammatory Composition Containing Pigment Extract from Morus alba L - Google Patents

Abstract

The present invention relates to an anti-inflammatory composition containing a pigment extract derived from Morus alba L. as an active ingredient, and more specifically, to an anti-inflammatory composition containing a pigment extract as an active ingredient obtained by a separation method comprising the steps of: extracting Morus alba L. by an ethyl acetate solvent; eluting the obtained insoluble fraction by a mixed solvent of hydrochloric acid and methanol; eluting the eluted fraction by the mixed solvent of hydrochloric acid and methanol; and separating the eluted fraction from a methanol aqueous solution by using high performance liquid chromatography. According to the present invention, the anti-inflammatory composition containing the pigment extract derived from Morus alba L. as an active ingredient is effective in inhibiting expression iNOS, phosphorylation of IκB-alpha, and activation of NF-κB related to inflammation, and has no concern of side effects compared to other existing synthetic drugs by using the natural figment extract, thereby being used as an anti-inflammatory composition.

Description

(Anti-inflammatory Composition Containing Pigment Extract from Morus alba L)

The invention mulberry (Morus The present invention relates to an antiinflammatory composition containing an alba L-derived pigment extract as an active ingredient. More particularly, the present invention relates to an antiinflammatory composition comprising an extract of olive oil with ethyl acetate, a step of eluting the insoluble fraction obtained with a mixed solvent of hydrochloric acid and methanol, Which comprises extracting the fractions obtained in step (a) with a mixture solvent of hydrochloric acid and methanol, and separating the eluted fractions in an aqueous methanol solution using fractional liquid chromatography, as an active ingredient .

Although the inflammatory response is a body defense mechanism for a variety of stimuli (Anderson et al., Pharmacol. Rev. 60: 311357, 2008), chronic inflammatory reactions are associated with various diseases including arthritis, hepatitis, septic shock and neuropathic disorders (Chung et < RTI ID = 0.0 > al ., Clin . Rheumatol . 26: 12281233, 2007). Macrophages play an important role in many inflammatory responses (Mayeux et al ., J. Toxicol . Environ . Health . 51: 415435, 1997), LPS is an inflammatory mediator such as nitric oxide (NO), prostaglandin E ₂ (PGE ₂ ), and tumor necrosis factor-α (TNF-α), interleukin-1 Of macrophages producing inflammatory cytokines (Takeuchi et < RTI ID = 0.0 > al ., J. Biol . Chem . 281: 2136221368, 2006). Inducible nitric oxide synthase (iNOS) promotes the formation of excessive nitric oxide that can evolve into inflammatory diseases (Nathan et al., Curr . Opin . Immunol . 3: 6570, 1991), cyclooxygenase- (COX-2) is induced by inflammatory stimulation and reaction for the synthesis of PGE ₂ (Yoon et al. al ., J. Biosci . Bioeng . 107: 429438, 2009). Thus, overexpression of NO and PGE ₂ by iNOS and COX-2 plays an important role in regulating the inflammatory response. NF-B (nuclear factor-kappa B) transcription factors are important regulators of inflammatory responses (Zhang et al al ., J. Clin . Invest . 107: 1319, 2001). In normal cells, the NF-κB inhibitory protein binds to IB and is present in the cytoplasm in an inactivated state. However, by various stimuli such as TNF-α and LPS, IκB protein is phosphorylated and degraded and NF -KB is activated and migrates into the nucleus (Ghosh et < RTI ID = 0.0 > al ., Nat. Rev. Immunol . 8: 837848, 2008). NF-κB translocated into the nucleus binds to the DNA binding sites that regulate transcription of the target gene and binds to inflammatory mediators such as iNOS, COX-2, NO, PGE ₂ , TNF-α and IL-1 Inducing the transcription of inflammatory cytokines (Lappas et < RTI ID = 0.0 > al ., Biol . Reprod . 67: 668673, 2002).

Audi is a Rosaceae banyanaceae plant, and is called heartburn, loss, nausea, and blackness in one room. It has high yield and high sugar content and is a promising variety of processed food. It is cultivated nationwide and has strong growth potential. In particular, anthocyanin, a pigment contained in audi, is a flavonoid-based pigment widely distributed in nature, and is known to have antioxidant and anti-inflammatory activity. With respect to the physiologically active functions of the audio frozen study on anti-inflammatory and antioxidant effect of the dried mulberry mulberry extract (Kim et al., Korean J. Medicinal Crop Sci . 6 (3): 204-209, 1998). However, studies on the physiological activity of audi have been conducted mainly on the form of crude extracts which have not been separated or purified, and it is extremely rare to separate and purify substances having physiologically active functions.

Therefore, the present inventors have established a protocol for assaying OD cells using LC-MS for the purpose of clarifying functional derivatives derived from osteoarthritis. To investigate their physiological activities, iNOS and COX-2 To confirm the anti-inflammatory effect of the dye-derived pigment extract, and the present invention was completed.

It is an object of the present invention to provide an anti-inflammatory composition containing an extract of a pigment derived from an oder as an active ingredient.

In order to achieve the above object, the present invention provides an anti-inflammatory composition comprising a coloring matter extract obtained by a separation method comprising the following steps as an active ingredient.

(a) extracting an oily product with an ethyl acetate solvent;

(b) eluting the insoluble fraction obtained in step (a) with a mixed solvent of hydrochloric acid and methanol;

(c) eluting the fraction eluted in the step (b) with a mixed solvent of hydrochloric acid and methanol; And

(d) separating the fraction eluted in the step (c) in an aqueous methanol solution using fractional liquid chromatography.

The antiinflammatory composition containing the dye-derived pigment extract according to the present invention as an active ingredient has an effect of inhibiting iNOS expression, inhibiting IκB-α phosphorylation and inhibiting NF-κB activation in relation to the inflammatory reaction, As compared with synthetic drugs, there is no concern about side effects, and therefore, they can be usefully used as an anti-inflammatory composition.

FIG. 1 shows a process of extracting a pigment from each of fractions of an audi using a multistage extraction method.
FIG. 2 compares the anti-inflammatory effects of the pigment extracts of each of the fractions of Audi. (A, B) and COX-2 (A, C) proteins by fractionation.
Fig. 3 compares the anti-inflammatory effects of the pigment extracts of each fraction of the OD4 MF4 fraction. (A, B) and COX-2 (A, C) proteins by fractionation.
FIG. 4 shows the anti-inflammatory effect according to the concentration of OD4 MF4-1 small fraction. The expression of iNOS (A, B) and COX-2 (A, C) proteins was analyzed by MF4-1 concentration.
FIG. 5 shows the anti-inflammatory effect according to the concentration of OD4 MF4-3 fraction. The expression of iNOS (A, B) and COX-2 (A, C) proteins was analyzed by MF4-3 concentration.
FIG. 5 shows the anti-inflammatory effects of the Oddi pigment extracts by investigating IκB-α phosphorylation inhibition and NF-κB activity of ODF MF4-1 and MF4-3 subfractions.
Fig. 6 is an analysis of the main substances of pigment extracts by liquid chromatography mass spectrometry (LC-MS) of OD4 MF4-3 small fractions.
Figure 7 compares the anti-inflammatory effect of the OD4 MF4-3 subfraction with C3G and C3R mixtures. The results of analysis of iNOS and COX-2 protein expression (B) of MF4-3, C3G and C3R mixture after quantification (A) of C3G and C3R contained in 50ug / ml of MF4-3 small fraction.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.

In the present invention, a methanol fraction was extracted using a multistage extraction method to isolate a substance having excellent anti-inflammatory effect among the coloring matters of Audi. Among the extracted fractions, fractions with excellent inhibition of iNOS expression were selected and a small fraction was extracted with methanol. Inhibition of IκB-α phosphorylation and inhibition of NF-κB activation of the fraction with the highest inhibitory effect on iNOS expression was confirmed, and it was confirmed that the extract of Oddi pigment was involved in the high signaling of the anti-inflammatory mechanism. Finally, in order to identify the major constituents of the ODI pigment extract fraction having excellent antiinflammatory effect, analysis of anti-inflammatory pigment substances using liquid chromatography mass spectrometry (LC-MS) Respectively.

In one aspect, the present invention relates to an anti-inflammatory composition containing a coloring matter extract obtained by a separation method comprising the following steps as an active ingredient.

(a) extracting an oily product with an ethyl acetate solvent;

(b) eluting the insoluble fraction obtained in step (a) with a mixed solvent of hydrochloric acid and methanol;

(c) eluting the fraction eluted in the step (b) with a mixed solvent of hydrochloric acid and methanol; And

(d) separating the fraction eluted in the step (c) in an aqueous methanol solution using fractional liquid chromatography.

Pigment extract of the present invention mulberry (Morus alba L), characterized in that it is extracted by a multistage extraction method with a solvent selected from the group consisting of water, ethyl acetate, methanol, hydrochloric acid, and mixtures thereof.

The coloring matter extract of the present invention can be obtained by using Tamarixetin 7-glucoside, Tamarixetin 3-galactoside, Taxifolin-hexoside, (±) - (+) -Taxifolin, malvidin 3-O- (6-O- (4-O-malonyl-alpha-ramanopyranosyl) -beta-glucopyranoside (4-O-malonyl-alpha-rhamnopyranosyl) -beta-glucopyranoside, Nathudaidin 3- (4-0-3-hydroxy-3-methylglutaroyl glucoside O-3-hydroxy-3-methylglutaroylglucoside), cyanidin 3-O-glucoside, cyanidin 3-rutinoside, acoseptrine, 5-O-feruloylquinic acid dimer, Malvidin 3-sophoroside-5-glucoside, 7-O-methyl-cyanoborohydride, (7-O-Methyl-cyanidin-3-O-galactoside) and Pigment A aglycone It characterized in that it comprises a play (Malvidin 4-vinylphenol)).

The anti-inflammatory composition containing the colorant extract of the present invention as an active ingredient is characterized by inhibiting the expression of iNOS, inhibiting IκB-α phosphorylation and activating NF-κB (nuclear factor kappa B) do.

The definitions of the main terms used in the description of the present invention and the like are as follows.

In the present invention, inflammation is one of biological tissue defense mechanisms against certain stimuli, and is a biologic defense mechanism to recover injured by various harmful stimuli to restore original state. Irritation of the inflammation, infection, or chemical and physical stimulation, and the process of inflammation can be divided into two types of acute and chronic inflammation. Acute inflammation is a short-term reaction within a few days. Plasma components and blood cells are involved in dephosphorylation by displaying a microcirculatory system. Chronic inflammation has a long duration, and tissue proliferation is seen.

In the present invention, nitric oxide is a substance that increases the production amount by nitric oxide synthase in inducing an intracellular inflammatory reaction, and is a molecule which is an index of an inflammatory reaction.

Herein, an enzyme involved in the production of prostaglandin, which is a protein involved in an inflammatory reaction, can be an indicator that the inflammatory response is proceeding, by increasing the level of intracellular COX-2 expression.

[Example]

Hereinafter, the present invention will be described in more detail by way of examples. It will be apparent to those skilled in the art that these embodiments are merely illustrative of the present invention and that the technical scope of the present invention is not limited to these embodiments.

Example 1 Extraction of Oddi Dye Substance by Fractionation Extraction

Ethyl acetate and methanol were used to extract the coloring matter of the olive oil. Methanol fraction was subdivided into four stages according to temperature and pH. Six fractions (MF1, MF2, MF3, MF4, MF5, and MF6) (Fig. 1).

Fraction 1 (MF1) was obtained by repeating the process of fractionating only the supernatant at 10 ° C with 10 ml of ethyl acetate twice. After washing with 10 ml of 0.1 M sodium acetate buffer and 10 ml of water, the ethyl acetate layer Fraction 2 (MF2) was prepared. To the ethyl acetate extract residue were added 1 ml of 2M HCl and 10 ml of methanol, and the supernatant was fractionated at 4 ° C. To the residue was added 10 ml of methanol, and the supernatant was fractionated at 4 ° C to obtain fraction 3 (MF3) . Fraction 4 (MF4) was extracted by reacting MF3 with methanol containing 0.6 M HCl at 60 ° C for 20 hours. The MF3 residue was reacted with 0.6 M HCl-containing methanol at 60 ° C for 20 hours, and the supernatant fraction was fractionated at 4 ° C to prepare a fraction 6 (MF6). The fraction 5 (MF5) Was reacted at 60 ° C for 20 hours, and then the supernatant was fractionated at 4 ° C.

Example 2: Small fraction of Audi coloring matter using Preparative-LC

MF4 sub-fractions from the fraction extracted in Example 1 were extracted.

The solvent was 0.1% formic acid and methanol, and a C18 column (DispoPack AT: 40 g, 25 μm) and a PDA detector (UV measurement wavelength: 280 nm, 360 nm, 400 nm) were used. The injection amount of the sample was adjusted to 10 ml and the flow rate to 25 ml / min. Solvent conditions were 0% methanol for 0-3 min, gradient from 0-100% methanol for 3-25 min, and 100% methanol for 25-30 min. A total of 6 subfractions (MF4-1: 3-7 min, MF4-2: 8-11 min, MF4-3: 12-15 min, MF4-4: 16-19 min, MF4-5: -23 min, MF4-6: 25-30 min).

Example  3: Fraction of the Audi coloring matter substance and Sub-fraction iNOS  And COX -2 protein expression analysis

3-1: The color of the material By fraction iNOS  And COX -2 protein expression analysis

The effects of iNOS and COX-2 protein expression on the six fractions extracted in Example 1 were analyzed.

RAW264.7 cells, a mouse Leukemia cell line, were stimulated with LPS (100 ng / ml) to induce inflammation, and the effect of each fraction (300 ug / ml) of ODi on iNOS and COX-2 protein expression was analyzed by western blotting and RT -PCR. ≪ / RTI >

As a result, it was confirmed that the MF4 fraction inhibited the expression of iNOS most effectively (Fig. 2).

3-2: The Sub-fraction iNOS  And COX -2 protein expression analysis

The effects of iNOS and COX-2 protein expression on six small fractions of the MF4 fraction extracted in Example 2 were analyzed.

The effect of the small fraction of MF4 (30 ug / ml) on iNOS and COX-2 protein expression was analyzed by Western blot and real-time RT-PCR.

As a result, it was confirmed that the MF4-1 and MF4-3 fractions inhibit the expression of iNOS most effectively (Fig. 3).

Example  4: MF4 Fractional  By concentration iNOS  And COX -2 Protein Expression Inhibitory Effect

In this example, the inhibitory effects of MF4-1 and MF4-3 on the inhibition of iNOS and COX-2 protein expression were analyzed.

RAW264.7 cells were stimulated with LPS (100 ng / ml) to induce inflammation and suppression of iNOS and COX-2 protein expression at concentrations of MF4-1 and MF4-3 fractions of 10, 30, 50, 100 and 300 ug / ml The effects were analyzed using Western blot and real-time RT-PCR.

As a result, it was confirmed that both MF4-1 and MF4-3 inhibited the expression of iNOS at a concentration of 30 ug / ml (FIGS. 4 and 5).

Example 5: Analysis of IκB-α phosphorylation and NF-κB activity of a small fraction of the Odi pigment extract

In this example, the effects of MF4-1 and MF4-3 on IκB-α phosphorylation and NF-κB activity were analyzed.

RAW264.7 cells were stimulated with LPS (100 ng / ml) to induce inflammation, phosphorylation and degradation of IκB-α at concentrations of MF4-1 and MF4-3 fractions of 10, 30, 50, 100 and 300 ug / ml Inhibition of NF-κB activation was analyzed.

As a result, MF4-1 did not show the phosphorylation and decomposition inhibitory effect of IκB-α (FIG. 6A), MF4-3 inhibited phosphorylation and decomposition of IκB-α and inhibited activation of NF-κB (FIG. 6B) . Thus, it was confirmed that the MF4-3 subfraction of the Odi extract inhibited the activation of NF-κB by inhibiting the LPS-induced inflammatory signaling.

Example  6: liquid chromatography mass spectrometry LC - MS ) Extracts of the Oddi pigment Small fraction (MF4-3)  Main material analysis

A small fraction (MF4-3) of the extract of Odidi pigment was filtered through a Millex (0.2 PTFE syringe filter, Japan) stage, concentrated under reduced pressure to remove the solvent, and then dissolved in methanol. Analysis was carried out using LC-MS / MS (Accela HPLC, LTQ-Velocity trap mass spectrometer, Thermo Fisher Scientific, San Hose, CA, USA) , Acquity, Waters, Milford, Mass., USA). The separation conditions were as follows: solvent B (0.1% formic acid in water) and solvent B (0.1% formic acid in acetonitrile) were fed at a flow rate of 0.3 mL / min. Was measured at 36 ° C and absorbance at 180-800 nm. MS information was collected at 100-1000 kDa for both positive and negative ion modes, the capillary temperature was adjusted to 275 ° C, and the source voltage was adjusted to 5 kV.

The results of the analysis are shown in FIG. 7, and the main substances of the Audi coloring matter extract are shown in the following table (Table 1).

Example  7: MF4 -3 Small fraction  Extract and C3G  And C3R  Mixture of iNOS  And COX -2 protein expression comparison

As a result of LC-MS analysis of 50 ng / ml MF4-3 fraction extract, 0.0324 mg / ml of Cyanidin 3-glucoside (C3G) and 0.0154 mg / ml of Cyanidin 3-rutinoside (C3R) (Fig. 8A).

In order to compare the anti-inflammatory effects of C3G and C3R, which are major anthocyanin constituents, with MF4-3, RAW264.7 cells were stimulated with LPS (100 ng / ml) to induce inflammation and C3G + C3R (X1: 0.0324 mg / ml The effect of inhibiting iNOS and COX-2 protein expression of 50 ng / ml MF4-3 and C3G + C3R (X5), C3G + C3R (X10) and 50 ng / ml MF4-3 was analyzed by Western blotting.

As a result, the 50 ng / ml MF4-3 pigment extract showed a similar level to the inhibition of iNOS expression of C3G + C3R (X10), confirming that the MF4-3 pigment extract of Audi showed excellent anti-inflammatory effect.

Claims (9)

An anti-inflammatory composition comprising a colorant extract obtained by a separation method comprising the following steps as an active ingredient:
(a) dissolving Odyssey ( Morus alba L) in ethyl acetate solvent to obtain a residue after two fractions of ethyl acetate supernatant;
(b) fractioning the insoluble residue obtained in step (a) with a mixed solvent of 1 ml of 2M hydrochloric acid and 10 ml of methanol, and then dividing the residue into 15 ml of methanol to elute a total of 25 ml of the supernatant fraction;
(c) eluting the fractions eluted in step (b) with methanol containing 0.6 M hydrochloric acid at 60 ° C for 20 hours to elute the fractions; And
(d) The fraction eluted in step (c) is purified by preparative liquid chromatography using 0.1% (v / v) formic acid and 0-100% (v / v) aqueous methanol solution, (V / v) methanol until 25 minutes, and 0% (v / v) methanol until 3 minutes, and 0-100% (v / v) methanol until 3-25 minutes, Separating the fraction from 12 to 15 minutes in an aqueous methanol solution.
delete delete delete delete delete delete The method according to claim 1, wherein the pigment extract is selected from the group consisting of tamarixetin 7-glucoside, tamarixetin 3-galactoside, taxifolin-hexoside, (±) -Taxifolin, malvidin 3-O- (6-O- (4-O-malonyl-alpha-ramanopyranosyl) -beta-glucopyranoside (Malvidin 3- O- (4-O-malonyl-alpha-rhamnopyranosyl) -beta-glucopyranoside, Nathudaidin 3- (4-0-3-hydroxy-3-methylglutaroyl glucoside 3- (4-O-3-hydroxy-3-methylglutaroylglucoside), cyanidin 3-O-glucoside, cyanidin 3-rutinoside, 5-O-feruloylquinic acid dimer, Malvidin 3-sophoroside-5-glucoside, 7-O Methyl-cyanidin-3-O-galactoside and Pigment A aglycone (erythrocyte-3-O-galactoside) 4-vinyl phenol (Malvidin 4-vinylphenol)) anti-inflammatory composition characterized in that it comprises a.
The anti-inflammatory composition according to claim 1, wherein the pigment extract inhibits the expression of an inductive nitric oxide enzyme (iNOS), inhibits IκB-α phosphorylation, and activates NF-κB (nuclear factor kappa B).

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