KR101227081B1 - Anti-inflammation Composition Using a phenylpropanoid derivative isolated from Sara quelpaertensis Nakai - Google Patents

Abstract

The present invention provides 3- O - p -coumaroyl- having NO production inhibitory activity, PGE ₂ production inhibitory activity, production inhibitory activity of inflammatory cytokine IL-6, iNOX production inhibitory activity, and / or COX-2 production inhibitory activity. An anti-inflammatory composition using 1- (4-hydroxy-3,5-dimethoxyphenyl) -1-O-β-D-glucopyranosylpropanol is disclosed.

Description

Anti-inflammation Composition Using a phenylpropanoid derivative isolated from Sara quelpaertensis Nakai}

The invention Jeju Sasa (Sara quelpaertensis It relates to an anti-inflammatory composition using a phenyl propanoid derivative isolated from Nakai). Specifically, a 3- O isolated from Cheju Sasa - p - one to Kumar-1- (4-hydroxy-3,5-dimethoxyphenyl) -1-O-β-D- glucopyranosyl-propanol (3- O -anti-inflammatory composition with p- coumaroyl-1- (4-hydroxy-3,5-dimethoxyphenyl) -1- O- β-glucopyranosylpropanol).

Inflammation is the defensive response of a living body to external infectious agents such as external physical and chemical stimuli, bacteria, fungi, viruses and allergens.

The inflammatory response is part of the innate immune response, and as in other animals, the innate immune response of humans begins by recognizing and attacking macrophages as non-self through a pattern of cell surfaces that are specific to the pathogen. During the inflammation reaction, plasma accumulates on the inflamed area, diluting the toxicities secreted by the bacteria, increasing the blood flow, and accompanied by symptoms such as erythema, pain, edema, and fever.

These inflammatory reactions involve a variety of biochemical events, in particular, cyclooxygenase (COX), which is associated with the nitric oxide synthase (NOS) and the biosynthesis of various prostaglandins, .

There are three isomers of NOS: endotoxin of bacteria such as calcium or camodanol-dependent eNOS (endothelial NOS), nNOS (neurogenic NOS), and lipopolysaccharide (IL-1β, TNF-α, IL There are iNOS (inducible NOS) induced by various inflammatory cytokines such as IL-6, IL-8 and IL-12 and produce NO from L-arginine.

NO produced by eNOS or nNOS plays an important role in maintenance of homeostasis by performing various physiological responses related to blood pressure control, neurotransmission, learning, memory, etc. However, NO produced by iNOS is associated with arthritis, sepsis, (Moncade S. et al, Pharmacol. Rev., 1991, 43, 109, Nature Medicine, 2001, 7, 1138; Mu, S., et al., Immunoprecipitation, , MM, J. Endotoxic Res. 7, p341, 2001).

The COX enzyme synthesizes PGG ₂ and PGH ₂ from arachidonic acid with hydroperoxidase (HOX) activity together with the function of COX. These compounds include PGE ₂ , PGF ₂ , PGD ₂ , prostacyclin and create a duplex thromboxane _{a 2 (thromboxane A2, TxA2)} . Among the functions of COX, the function of PGH synthase is involved in the pain and inflammation reaction through synthesis of PGE ₂ .

There are two subtypes of COX and COX-1 is always expressed in most tissues, whereas COX-2 is rapidly induced by inflammatory cytokines and plays an important role in the inflammatory response.

Therefore, NO, PGE ₂ , Production inhibitors such as inflammatory cytokines, iNOS inhibitors, and COX-2 inhibitors may be used as therapeutic agents for inflammatory diseases.

The present invention relates to a method for producing NO, PGE ₂ , 3- O - p -coumaroyl-1- (4-hydroxy-3), a phenylpropanoid derivative isolated from Jeju jejudae having iNOS and COX-2 inhibitory activity with inhibitory activity against inflammatory cytokines IL-6 , 5-dimethoxyphenyl) -1-O-β-D-glucopyranosylpropanol is disclosed.

An object of the present invention is to provide an anti-inflammatory composition using 3- O - p -coumaryl-1- (4-hydroxy-3,5-dimethoxyphenyl) -1-O-β-D-glucopyranosylpropanol. To provide.

Other and further objects of the present invention will be described below.

The present invention, as confirmed in the following Examples and Experimental Example, 3- O - p -coumaroyl-1- (4-hydroxy-3,5-dimethol, which is a phenylpropanoid derivative isolated from Jeju jeokdae By confirming that oxyphenyl) -1-O-β-D-glucopyranosylpropanol inhibits NO production, inhibits the production of the inflammatory cytokines IL-6 and PGE ₂ , and has iNOS and COX-2 inhibitory activity. It is completed.

3- O - p -coumaroyl-1- (4-hydroxy-3,5-dimethoxyphenyl) -1- O- β-D-glucopyranosylpropanol, which is the phenylpropanoid derivative, is the leaves of Jeju The structure and the name of IUPAC can be found in [Formula 1] below as separated from, and described in Bull . Korean Chem . Soc . 2009 , Vol. 30, No. 8 1729 ~ 1732]. The document is considered part of this specification.

Therefore, the anti-inflammatory composition of the present invention is represented by the following 3- O - p -coumaryl-1- (4-hydroxy-3,5-dimethoxyphenyl) -1-O-β-D- It is characterized by comprising glucopyranosylpropanol as an active ingredient.

[Formula 1]

The anti-inflammatory composition of the present invention is an effective ingredient thereof, as described in Bull . Korean Chem . Soc. 2009 , Vol. 30, No. 8 1729 ~ 1732, the methanol extract of Jeju jejugwa containing the compound of [Formula 1], the ethyl acetate fraction of the methanol extract, the column chromatography of the fraction and the like may be included.

In the present specification, the term "active ingredient" means a component that can exhibit activity alone or in combination with a carrier which is not active in itself.

As used herein, "anti-inflammatory" is meant to include the improvement, treatment, inhibition or delay of the onset of an inflammatory disease as defined below.

In the present specification, the above-mentioned "inflammatory disease" is defined as any state specified by a local or systemic bio-defense reaction against external physical or chemical stimulation or infection of an external infectious agent such as bacteria, fungi, viruses, . This reaction is enzyme secretion (e.g., iNOS, COX-2, etc.) activation, release of inflammatory mediators (e. G., NO, TNF-α, IL -6, IL-1β, PGE 2 related to various inflammatory mediators and the immune cells ), Bodily fluid infiltration, cell migration, and tissue destruction, and manifest externally by symptoms such as erythema, pain, edema, fever, deterioration or loss of specific function of the body. The inflammatory disease may be acute, chronic, ulcerative, allergic or necrotic, so long as it is included in the definition of inflammatory diseases as described above, it may be acute, chronic, ulcerative, allergic, Whether it is necrotic or not. Specifically, the inflammatory diseases include asthma, allergic and non-allergic rhinitis, chronic and acute rhinitis, chronic and acute gastritis or enteritis, ulcerative gastritis, acute and chronic nephritis, acute and chronic hepatitis, chronic obstructive pulmonary disease, pulmonary fibrosis Irritable bowel syndrome, inflammatory pain, migraines, headache, back pain, fibromyalgia, fascia disease, viral infections (eg, type C infections), bacterial infections, fungal infections, burns, surgical or dental wounds, Prostaglandin E excess syndrome, atherosclerosis, gout, arthritis, rheumatoid arthritis, ankylosing spondylitis, Hodgkin's disease, pancreatitis, conjunctivitis, irisitis, scleritis, uveitis, dermatitis (including atopic dermatitis), eczema, multiple sclerosis, etc. Will be included.

In another aspect of the present invention, 3- O - p -coumaroyl-1- (4-hydroxy-3,5-dimethoxyphenyl) -1-O-β-D-glucopyranosylpropanol is an active ingredient. It relates to an iNOS inhibitor composition comprising a.

As mentioned above, inhibition of iNOS can inhibit the production of NO leading to improvement of inflammatory disease.

The following experimental example of the present invention shows that 3- O - p -coumaroyl-1- (4-hydroxy-3,5-dimethoxyphenyl) -1-O-β-D-glucopyranosylpropanol is represented by NO. It has been shown to inhibit production and to inhibit the production of iNOS and to inhibit the production of cytokines known to induce the expression of iNOS.

As used herein, "iNOS inhibition" is meant to include inhibition of expression of iNOS gene and inhibition of iNOS production, and meaning including inhibition and / or reduction of NO production whatever the intermediate mechanism.

In another aspect, the present invention is effective for 3- O - p -coumaroyl-1- (4-hydroxy-3,5-dimethoxyphenyl) -1-O-β-D-glucopyranosylpropanol It relates to a COX-2 inhibitor composition comprising as a component.

As mentioned above, inhibition of COX-2 may inhibit the production of PGE ₂ resulting in improvement of inflammatory disease.

The following experimental example of the present invention is a 3- O - p -coumaroyl-1- (4-hydroxy-3,5-dimethoxyphenyl) -1-O-β-D-glucopyranosylpropanol PGE ₂ It has been shown to inhibit the production of and inhibit the production of COX-2, and to inhibit the production of cytokines known to induce the expression of COX-2.

As used herein, the term "COX-2 inhibition" is meant to include inhibition of expression of COX-2 gene and inhibition of COX-2 production, and meaning including inhibition and / or reduction of the production of PGE ₂ whatever the intermediate mechanism. to be.

Meanwhile, the composition of the present invention (i.e., anti-inflammatory composition, iNOS inhibitor composition, and COX-2 inhibitor composition; refers to the same as below) exhibits an improvement activity of an inflammatory disease in which the active ingredient is intended to be treated according to the use, formulation, formulation purpose, and the like. It may be included in any amount (effective amount) as far as possible, and a typical effective amount will be determined within the range of 0.001% to 15% by weight based on the total weight of the composition. The term "effective amount" as used herein refers to the amount of an effective ingredient capable of inducing the improvement, treatment, or suppression / delay of the development of such pathological symptoms in a mammal, preferably a human, to which it is applied. Such effective amounts can be determined experimentally within the ordinary skill of those skilled in the art.

Subjects to which the compositions of the invention can be applied (prescribed) are mammals and humans, in particular humans.

The composition of the present invention can be used as a pharmaceutical composition in a specific embodiment.

The pharmaceutical composition of the present invention includes oral formulations (tablets, suspensions, granules, emulsions, capsules, syrups, etc.), parenteral formulations (sterilized), including pharmaceutically acceptable carriers, excipients, etc., in addition to the active ingredients thereof. Aqueous or oily suspensions for injection), topical formulations (solutions, creams, ointments, gels, lotions, patches) and the like.

As used herein, "pharmaceutically acceptable" means that the subject of application (prescription) does not have more toxicity (adequately low toxicity) to which the subject of application (prescription) is adaptable without inhibiting the activity of the active ingredient.

Examples of pharmaceutically acceptable carriers include lactose, glucose, sucrose, starch (such as corn starch, potato starch, etc.), cellulose, derivatives thereof (such as sodium carboxymethyl cellulose, ethylcellulose, etc.) malt, gelatin, talc, solids Lubricants (e.g. stearic acid, magnesium stearate, etc.), calcium sulfate, vegetable oils (e.g. peanut oil, cottonseed oil, sesame oil, olive oil, etc.), polyols (e.g. propylene glycol, glycerin, etc.), alginic acid, emulsifiers (e.g. TWEENS), wetting agents (e.g. Sodium lauryl sulfate), colorants, flavoring agents, tableting agents, stabilizers, antioxidants, preservatives, water, saline, phosphate buffer solutions and the like. The carrier may be selected from one or more of suitable pharmaceutical formulations according to the formulation of the pharmaceutical composition of the present invention.

Excipients may be selected and used according to the formulation of the pharmaceutical composition of the present invention, for example, when the pharmaceutical composition of the present invention is prepared with an aqueous suspending agent, suitable excipients are sodium carboxymethyl cellulose, methyl cellulose, hydropropylmethylcellulose And suspending agents and dispersing agents such as sodium alginate and polyvinylpyrrolidone. Suitable excipients when prepared from injection solutions include Ringer's solution, isotonic sodium chloride, and the like.

The pharmaceutical compositions of the present invention may be administered orally or parenterally, and may optionally be administered topically, as in the case of atopic dermatitis compositions.

The daily dose of the pharmaceutical composition of the present invention is usually 0.001 to 150 mg / kg body weight, and may be administered once or several times. However, since the dosage of the pharmaceutical composition of the present invention is determined in view of various related factors such as route of administration, age, sex, weight, and patient's severity of the patient, the dose is limited in any aspect to the scope of the present invention Should not be understood to be.

The composition of this invention can be grasped | ascertained as a food composition in another specific aspect.

The food composition of the present invention may be prepared as a dietary supplement, a special nutritional supplement, a functional beverage, and the like.

The food composition of the present invention includes not only the active ingredient, but also sweeteners such as corn syrup solids, honey, sucrose, fructose, lactose and maltose, fruits such as apples, lemons and citrus fruits, green tea leaves, round stalks, and large leaves. Flavoring agents obtained from tea, catechins, retinol, ascorbic acid, tocopherol and other biologically active ingredients, calcium, magnesium, chromium, cobalt, copper, fluoride and the like may also be added.

In addition, herbal medicines may be added to enhance flavor and palatability and to add other functionalities (such as prevention of arthritis or osteoporosis). Herbal medicines that may be added include scavenger extract, tofu extract, fast extract, antler extract, safflower extract, earthworm Extracts, Sukjihwang extract, Tortoiseshell extract, Cornus extract, Goji berry extract, Licorice extract, Angelica extract, brown root extract, Gangjinhyang extract, Haphwanpi extract, mountain rump muscle extract, lump extract, red ginseng extract and the like can be exemplified.

As described above, the present invention can provide an anti-inflammatory composition.

The anti-inflammatory composition of the present invention may be commercialized as a drug or food.

1 shows that 3- O - p -coumaryl-1- (4-hydroxy-3,5-dimethoxyphenyl) -1-O-β-D-glucopyranosylpropanol has NO production inhibitory activity Results show no cytotoxicity.
FIG. 2 shows IL-6, wherein 3- O - p -coumaryl-1- (4-hydroxy-3,5-dimethoxyphenyl) -1-O-β-D-glucopyranosylpropanol is an inflammatory cytokine The results show that the production of PGE ₂ is inhibited.
Figure 3 Expression of iNOS and COX-2 of 3- O - p -coumaryl-1- (4-hydroxy-3,5-dimethoxyphenyl) -1-O-β-D-glucopyranosylpropanol Results show inhibitory activity.

Hereinafter, the present invention will be described with reference to Examples. However, the scope of the present invention is not limited to these examples.

< Example  1> Sample and other materials

The sample (ESQ10) was prepared from Jeju Osamu Osamu identified as 3- O - p -coumaryl-1- (4-hydroxy-3,5-dimethoxyphenyl) -1-O-β-D-glucopyranosylpropanol. Sasa quelpaertensis) fractions of leaf extracts (Bull. Korean Chem . Soc . 2009 , Vol. 30, No. 8 1729 ~ 1732) was used from Jeju National University.

DMEM (Dulbecco's Modified Eagle's Medium) medium and fetal bovine serum (FBS) used for cell passage were purchased from Gibco (Grand Island, NY, USA), and lipopolysaccharide (LPS, E. coli serotype 0111: B4) was obtained from sigma ( St. Louis, MO). The kit for enzyme-linked immunosorbent assay (ELISA) of IL-6 was purchased from BD Bioscience (San Diego, CA, USA) and the PGE ₂ ELISA kit was purchased from R & D systems (Minneapolis, MN, USA). Antibody against inducible NOS (iNOS) for Western blot was purchased from Calbiochem (San Diego, Calif., USA) and COX-2 from BD Bioscience. All reagents used sigma grade reagents.

< Example  2> Cell culture

RAW 264.7 cells, a Murine macrophage cell line, were distributed from Korean Cell Line Bank (Seoul, Korea) and DMEM medium containing penicillin (100 unit / mL), streptomycin (100 ㎍ / mL) and 10% fetal bovine serum (FBS) Incubated at 37 ℃, 5% CO ₂ incubator, and passaged once every 2-3 days.

< Example  3> Cytotoxicity Assessment

Sample (ESQ10) cytotoxicity was tested using MTT assay. Simultaneous treatment of samples (ESQ10) and LPS (1 μg / mL) in RAW 264.7 cells (1.0x10 ⁵ cells / mL) and incubated for 24 hours, 50 μl (2 mg / mL) MTT (Sigma, MO, USA) solution ) And react for 4 hours. The supernatant was completely removed, 200 μl of dimethylsulfoxide (DMSO: Sigma, MO, USA) was added to completely dissolve the precipitate, and the absorbance was measured at 540 nm using a microplate reader. The average absorbance value of each sample group was obtained and compared with the absorbance value of the control group, the degree of toxicity was evaluated.

The results are shown in FIG. 1. Referring to Figure 1 it can be seen that the sample (ESQ10) does not show a particular cytotoxicity at all treatment concentrations.

< Example  4> Nitric oxide  ( NO ) Measure

RAW 264.7 cells (1.5x10 ⁵ cells / mL) were incubated 18 hours ago and then incubated for 24 hours with simultaneous treatment of sample (ESQ10) and LPS (1 μg / mL). It was measured in the form of NO ₂ ^- present. 100 μl of the cell culture supernatant and the same amount of griess reagent [1% (w / v) sulfanilamide, 0.1% (w / v) naphylethylenediamine in 2.5% (v / v) phosphoric acid] Absorbance was measured at 540 nm using an ELISA reader. Standard concentration curves were obtained by serial dilution of sodium nitrite (NaNO ₂ ) (1-100 uM).

The results are shown together in FIG. 1. Referring to FIG. 1, it can be seen that the sample ESQ10 suppresses NO production in a concentration-dependent manner.

< Example  5> PGE ₂ Wow IL Quantification of -6

RAW 264.7 cells (2.5 × 10 ⁵⁾ , a Murine macrophage cell line cells / mL) were incubated 18 hours ago and co-treated with sample (ESQ10) and LPS (1 μg / mL) for 24 hours. After 24 hours, the contents of PGE ₂ and IL-6 were measured with the supernatant obtained by centrifugation (12,000 rpm, 3 min) of the culture medium. ELISA kit was used for quantification. R ² of the standard curve for the standard The value was 0.99 or more.

The results are shown in Figure 2, it can be seen that the sample (ESQ10) inhibits the production of PGE ₂ and IL-6 in a concentration-dependent manner.

< Example  6> Western blot analysis  - iNOS  And COX -2 quantification

RAW 264.7 cells (5.0X10 ⁵ cells / mL) were incubated 18 hours before, and then treated with sample (ESQ10) and LPS (1 μg / mL) for 24 hours. The cells were washed twice with PBS (phosphate buffered saline) and then 200 μl of lysis buffer [50 mM Tris-HCl (pH7.5), 150 mM NaCl, 1% Nonidet P-40, 2 mM EDTA, 1 mM EGTA, 1 mM NaVO ₃ , 10 mM NaF, 1 mM dithiothreitol, 1 mM phenylmethylsulofonyl fluoride, 25 μg / mL aprotinin, 25 μg / mL leupeptin] were added and lysed at 4 ° C. for 30 minutes to 1 hour, followed by 15 at 15,000 rpm. Centrifuged for a minute to remove cell membrane components and the like. Protein concentration was quantified using the Bio-Rad Protein Assay Kit by standardizing BSA (bovine serum albumin). 20-30 μg of lysate was denatured by 10-12% mini gel SDS-PAGE (Poly Acrylamide Gel Electrophoresis) and transferred to PVDF membrane (BIO-RAD, HC, USA). Membrane blocking was overnight in TTBS (TBS + 0.1% Tween 20) solution containing 5% skim milk. Antibodies to confirm the expression level of iNOS and COX-2 were diluted anti-rabbit iNOS (1: 2500) and anti-mouse COX-2 (1: 2500) in TTBS solution and reacted at room temperature for 2 hours, and then TTBS Washed 4 times. As a secondary antibody, HRP (Horse Radish Peroxidase) conjugated anti-rabbit IgG and anti-mouse IgG (Vector Laboratiories, Burlingame, USA) were diluted 1: 5000 and reacted at room temperature for 30 minutes, followed by 4 times with TTBS. Clean. After completion of the reaction, the membrane was reacted with ECL substrate (Intron Biotechnology, Inc, Korea) for 1 minute and then photosensitive on X-ray film.

The results are shown in FIG. 3. In the case of iNOS it can be seen that the production is inhibited in a concentration-dependent manner, and in the case of COX-2 it can be seen that the production is generally suppressed similar to the quantitative result of PGE ₂ of FIG.

Statistical processing

The experimental results show the data of three or more independent experiments as mean ± standard error. The statistical significance test between the groups was performed using student's t-test analysis.

Claims (6)

3- O - p -coumaryl-1- (4-hydroxy-3,5-dimethoxyphenyl) -1-O-β-D-glucopyranosylpropanol, a phenylpropanoid derivative isolated from Jeju jeokdae Anti-inflammatory composition comprising as an active ingredient.
The method of claim 1,
The anti-inflammatory refers to amelioration, treatment, inhibition of onset or delayed on the inflammatory disease,
The inflammatory diseases include asthma, allergic and non-allergic rhinitis, chronic and acute rhinitis, chronic and acute gastritis or enteritis, ulcerative gastritis, acute and chronic nephritis, acute and chronic hepatitis, chronic obstructive pulmonary disease, pulmonary fibrosis, irritability Bowel Syndrome, Inflammatory Pain, Migraine, Headache, Back Pain, Fibromyalgia, Fascia Disease, Viral Infection, Bacterial Infection, Fungal Infection, Burn, Surgical or Dental Surgery, Prostaglandin E-Over Syndrome, Atherosclerosis Atherosclerosis, gout, arthritis, rheumatoid arthritis, ankylosing spondylitis, Hodgkin's disease, pancreatitis, conjunctivitis, irisitis, scleritis, uveitis, dermatitis, atopic dermatitis, eczema and multiple sclerosis.
The method according to claim 1 or 2,
The composition is an anti-inflammatory composition, characterized in that the pharmaceutical composition.
3- O - p -coumaryl-1- (4-hydroxy-3,5-dimethoxyphenyl) -1-O-β-D-glucopyranosylpropanol, a phenylpropanoid derivative, is included as an active ingredient. A composition for improving an inflammatory disease. delete delete

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