KR20130046138A - Composition for preventing or treating inflammation comprising anethum graveloens extracts - Google Patents

Abstract

PURPOSE: A composition containing an Anethum graveloens extract for preventing or treating inflammatory diseases is provided to suppress NO generation and nitric oxide synthase expression, and to ensure anti-inflammatory effect. CONSTITUTION: A composition for preventing or treating inflammatory diseases contains an Anethum graveloens flower extract as an active ingredient. The extract is prepared using water, C1-C6 alcohols, or a mixture thereof. The extract is also an organic solvent fraction of a low carbon number alcohol extract of Anethum graveloens. The inflammatory diseases are rheumatoid arthritis or asthma. The composition is manufactured in the form of a capsule, a liquid, an injection, a soft capsule, a granule, or a tablet. [Reference numerals] (AA) Nitrogen oxide(uM)

Description

Composition for preventing or treating inflammation comprising Anethum graveloens extracts

The present invention provides a composition for the prevention or treatment of inflammatory diseases, including the small fennel extract, which is used for the prevention or treatment of inflammatory diseases by identifying that the fennel flower extract inhibits nitric oxide production and the expression of nitric oxide synthase. It is about.

The causes of chronic inflammatory diseases (eg, rheumatoid arthritis, asthma) arise from immune abnormalities in the body. For example, when the body's immune system removes them from the invasion of bacteria or viruses, it attacks the joints or parts of the body, causing symptoms of rheumatoid arthritis. About 1% of the country's population is serious enough to have rheumatoid arthritis, and further complications. Bronchial asthma is a disease that is accompanied by coughing, shortness of breath, tightness of the chest, and bronchial spasms. If the symptoms are severe, it is a condition that replaces all of the treatment with treatment such as bronchodilators temporarily. In recent years, the number of patients is increasing due to environmental pollution.

So far, nonsteroidal analgesic anti-inflammatory drugs and corticosteroids have been used for pain relief symptoms of chronic inflammatory diseases, but when taken chronically, they cause serious side effects such as gastrointestinal disorders and gastric ulcers. Therefore, the development of new medicines that can treat inflammatory diseases is urgent and urgent.

1. Republic of Korea Patent Publication No. 2011-0046210, 2011.05.04 2. United States Patent Application Publication No. 2008-0166435, 2008.07.10

Eun Mi CHoi et al. Fitoterapia, vol. 75 (6), pp. 557-565, 2004.09.

It is an object of the present invention to provide a composition which effectively prevents or treats an inflammatory disease using a natural extract without side effects.

Another object of the present invention to provide a dietary supplement comprising a natural extract as an active ingredient that can effectively prevent or improve inflammatory diseases.

In order to achieve the above object, the present invention is a small fennel ( Anethum) graveolens ) Provides a composition for the prevention or treatment of inflammatory diseases comprising a flower extract as an active ingredient.

The invention also relates to small fennel ( Anethum) graveolens ) Provides a dietary supplement for the prevention or improvement of inflammatory diseases, including flower extract as an active ingredient.

Since the fennel extract of the present invention exhibits nitric oxide (NO) and nitric oxide synthase inhibitory action, it can be effectively used for preventing, improving or treating inflammatory diseases, particularly rheumatoid arthritis, asthma, and the like without side effects.

1 is a graph showing the inhibitory effect of the small fennel extract of the present invention on nitric oxide production induced by lipopolysaccharide (LPS).
Figure 2 is a graph showing the effect of the small fennel extract of the present invention on nitric oxide synthase induced by LPS.
Figure 3 is a graph showing the effect of the small fennel extract of the present invention on NF-κB activity induced by LPS.
Figure 4 is a graph showing the cell viability of the fennel extract of the present invention.

EMBODIMENT OF THE INVENTION Hereinafter, the structure of this invention is demonstrated concretely.

The present inventors have sohoehyang (Anethum with new candidates that may be developed in inflammatory disease treatment graveloens ) was selected and its anti- inflammatory activity was investigated.

Sohoehyang (Anethum graveloens) shall multiply by seed or rhizome herbaceous perennial belonging to the fennel (fennel, Foeniculum vulgare) with yearly plant of Apiaceae (Umbelliferae) and the wild country around the country. Since small fennel has a unique scent throughout the plant, flowers, leaves, stems, and seeds are used as herbs, and the scent of seeds is particularly strong. Seeds of small fennel have effects on the central nervous system, soothing, stimulating mucous membranes to help secretion of the glands such as stomach, intestines, throat, and breast, digestive disorders, sputum, pain relief, and pain relief It is written in Chinese medicine. As constituents of small fennel, essential oils in seeds, phenolic compounds in flowers, flavonoids and proanthocyanidins have been reported.

Since the anti-inflammatory effect of small fennel has not been reported, the present inventors earnestly conducted a study on small fennel. As a result, the ethanol extract of the small fennel flower showed anti-inflammatory effect by inhibiting the expression of nitric oxide and nitric oxide synthase. It confirmed and completed this invention.

Thus, the present invention is a small fennel ( Anethum) graveolens ) Provides a composition for the prevention or treatment of inflammatory diseases comprising a flower extract as an active ingredient.

The composition for the prevention or treatment of inflammatory diseases according to the present invention may be prepared as a pharmaceutical preparation for the prevention or treatment of inflammatory diseases, in particular rheumatoid arthritis or asthma.

The pharmaceutical preparation has no side effects and uses very high safety as compared to conventional organic synthetic drugs by using a natural material separated from the small fennel flowers.

The separation method of the small fennel flower extract of the present invention is not particularly limited, but after extraction at 90-100 ° C. with water or a lower alcohol having 1 to 6 carbon atoms or a mixed solvent thereof, concentrated under reduced pressure or lower alcohol having 1 to 6 carbon atoms, In particular, the ethanol extract may be further fractionated with various organic solvents such as hexane, chloroform, ethyl acetate or butanol, and may be further purified by various chromatography after organic solvent fractionation.

The small fennel flower extract of the present invention shall include any one of all extracts, fractions and purified products obtained in each step of extraction, fractionation and purification, dilutions or concentrates thereof or dried products thereof. Preferably, ethanol extracts of small fennel flowers are used.

The small fennel flower extract has an anti-inflammatory effect by inhibiting nitric oxide production and nitric oxide synthase expression.

According to a specific embodiment of the present invention, when inducing an inflammatory response to macrophages using lipopolysaccharide, various inflammatory mediators such as nitric oxide (NO) are released, the fennel flower extract is concentration-dependent By reducing the concentration of nitric oxide in a manner that has the effect of inhibiting the inflammatory response induced by lipopolysaccharide in macrophages. In addition, expression of nitric oxide synthase (iNOS) is also reduced in a concentration dependent manner. In other words, the small fennel flower extract exhibits an anti-inflammatory effect by reducing the concentration of nitric oxide by inhibiting the expression of nitric oxide synthase. In addition, small fennel flower extracts inhibit the activity of NF-κB in a concentration dependent manner. It can be seen that the small fennel flower extract prevents the degradation of IkB and directly inhibits the activity of NF-κB.

In addition, the small fennel flower extract did not affect the cell viability in the cell viability measurement experiment, it was confirmed that there is no toxicity.

In addition, the composition for preventing or treating inflammatory diseases of the present invention may further comprise a pharmaceutically acceptable carrier.

Such pharmaceutically acceptable carriers include carriers and vehicles commonly used in the medical field and specifically include ion exchange resins, alumina, aluminum stearate, lecithin, serum proteins (e.g., human serum albumin), buffer substances Water, salts or electrolytes (e.g., protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride and zinc salts), colloidal silicon dioxide But are not limited to, silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose based substrate, polyethylene glycol, sodium carboxymethylcellulose, polyarylate, wax, polyethylene glycol or wool.

In addition, the composition of the present invention may further include a lubricant, a wetting agent, an emulsifier, a suspending agent, or a preservative in addition to the above components.

In one embodiment, the composition according to the present invention may be prepared as an aqueous solution for parenteral administration, preferably a buffer solution such as Hank's solution, Ringer's solution or physically buffered saline Can be used. Aqueous injection suspensions may contain a substrate capable of increasing the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran.

The composition of the present invention may be administered systemically or locally, and may be formulated into a formulation suitable for such administration by known techniques. For example, upon oral administration, it may be admixed with an inert diluent or edible carrier, sealed in a hard or soft gelatin capsule, or pressed into tablets. For oral administration, the active compound may be mixed with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.

Various formulations for injection, parenteral administration and the like can be prepared according to techniques known in the art or commonly used techniques.

Suitable dosages of the compositions of the present invention are appropriately selected depending on the absorption, excretion rate, active age and weight of the patient, sex and condition, the severity of the disease to be treated, etc., in general, from 10 mg per day to adults 5,000 mg may be administered 1 to 3 times, and the amount may be increased or decreased depending on the weight, sex, age, and degree of disease of the patient.

The composition of the present invention may be administered orally or parenterally, and when administered parenterally, may be administered by intravenous infusion, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, or the like. It is preferable that the route of administration of the composition of the present invention is determined according to the type of the disease to be applied.

The compositions of the present invention may be prepared in unit dosage form by formulating with a pharmaceutically acceptable carrier and / or excipient according to methods which can be easily carried out by those skilled in the art. It can be prepared by incorporation into a multi-dose container. In this case, the formulation may be in the form of injections, solutions, powders, granules, tablets, soft capsules, or capsules in the form of solutions, suspensions or emulsions in an oil or aqueous medium, and may further include a dispersing or stabilizing agent.

The present invention also provides a method for treating inflammatory disease in an animal comprising administering to the individual a composition for preventing or treating an inflammatory disease comprising a pharmaceutically effective amount of a small fennel extract.

Since the pharmaceutical composition and the method of administration used in the method of treating the inflammatory disease have been described above, the contents in common between the two are omitted in order to avoid excessive complexity of the present specification.

On the other hand, the individual capable of administering the pharmaceutical composition for preventing or treating the inflammatory disease includes all animals. For example, it may be an animal other than a human such as a dog, a cat, and a mouse.

The invention also relates to small fennel ( Anethum) graveolens ) relates to a dietary supplement for the prevention or improvement of inflammatory diseases comprising an ethanol extract of a flower as an active ingredient.

The "health supplement" is a food prepared by adding the fennel flower extract to food materials such as beverages, teas, spices, gums, confections, or the like, encapsulated, powdered, suspensions, etc. It means that there is, unlike the general medicine has the advantage that there is no side effect that can occur when taking a long-term use of the drug as a raw material. Since the dietary supplement of the present invention thus obtained can be consumed on a daily basis, a prophylactic effect of high chronic inflammatory diseases can be expected and is very useful.

In the dietary supplement of the present invention, the addition amount of the small fennel flower extract can not be defined uniformly depending on the type of the dietary supplement, but may be added in a range that does not impair the original taste of the food, usually with respect to the target food It is 0.01-50 weight%, Preferably it is the range of 0.1-20 weight%. In the case of food in the form of granules, tablets or capsules, it is usually added in the range of 0.1 to 100% by weight, preferably 5 to 100% by weight.

Hereinafter, the present invention will be described in more detail with reference to Examples of the present invention, but the scope of the present invention is not limited by the following Examples.

Example 1 Preparation of Small Fennel Extract

Sohhoe was cultivated by the RDA's Good Agricultural Practice (GAP), and was harvested in 2009 in Chungcheongbuk-do Negong (GPS: E 128 ° 62´ N 36 ° 56´). Flower samples were extracted with 99% ethanol and used as test material.

Example 2 Screening for Inflammatory Defense of Macrophages

In the process of creating a substance with macrophage inflammatory defense activity from natural products, the first requirement is the establishment of a suitable screening method to search for substances having such activity. Macrophages of rats known to be physiological and biochemically similar to human macrophages have been widely used as search systems in order to find substances having inflammatory defense activity from natural products. In order to detect macrophage protection activity in natural products, the natural macrophages are treated with natural products, and after a certain time, endotoxins are treated to measure the degree of protective effect when artificially inflamed.

In the present invention, lipopolysaccharide (LPS) was used to induce inflammation in macrophages. LPS is a major component of the Gram-negative bacterial cell wall, and it binds to the plasma LPS binding protein (LPB) in the cytoplasm and moves to the phospholipid of the cell membrane to bind to CD14 present on the surface of the macrophage, or 95kDa in which the LPS itself is present on the cell membrane. , 80kDa represent the inflammatory response in conjunction with a protein such as (Hewett, JA and Roth, RA :... Hepatic and extrahepatic pathobiology of bacterial lipopolysaccharides Pharmacol Rev 45, 382-411 (1993)). The binding of LPS to the receptor stimulates intracellular G1 protein and results in tumor nacrosis factor (TNF-α), interleukin- and TNF-α signaling from cells through signaling systems of mitogen activated protein kinase (MAPK). Various inflammatory mediators such as cytokines such as 1 (IL-1), IL-6, prostanoids, leukotriens and nitric oxide (NO) do.

In this experimental example, to determine the anti-inflammatory effects of small fennel extract in macrophages, the protective effect against LPS was measured at the quantitative measurement of nitric oxide and at the protein level of nitric oxide synthase (iNOS), and the activity of NF-κB was measured. It was. In addition, cell viability was measured to observe the toxicity of the fennel extract. The data shown in this experiment was analyzed using a pharmacologic calculation program. Significance between the various treatment groups was tested by one way analysis of variance (ANOVA) followed by Newmann-Kelus test (** p <0.01).

(1) RAW264.7 cell culture, mouse macrophage line

The cell used to test the inflammatory response was Raw 264.7, a macrophage extracted from male rats, which was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Raw 264.7 cells were treated at 37 ° C., 5% in Dulbecco's Modified Eagle's Medium (DMEM, Sigma-Aldich, St. Louis, MO, USA) medium containing 10% bovine serum, 100 U / mL penicillin and 100 mg / mL streptomycin. It was incubated under the conditions of CO ₂ .

(2) Determination of the effect of small fennel extract on nitric oxide production

Raw 264.7 cells were planted in 24-well plates at 1 × 10 ⁶ cells / mL and incubated for 4 hours. In each well of a 24-well plate a small fennel extract was added at each concentration (5, 25, 50, or 100 μg / mL) and incubated in the incubator for 30 minutes. Thereafter, LPS was put in each well to a concentration of 100 ng / mL, and then incubated for 18 hours in an incubator. 100 mL of each supernatant of each well was dispensed into a 96-well plate. 100 mL of Griess reagent (1% sulfanilamide, 0.1% N- (1-naphthyl) ethylenediamine dihydro-chloride, 2.5% phosphoric acid) The same was put in each well of a 96-well plate, and reacted at room temperature for 5 minutes. Absorbance was measured at 550 nm using a microplate reader. At this time, the concentration was measured using sodium nitrite as a standard curve. The results are shown in FIG.

Raw 264.7 cells induced by LPS were confirmed to increase the concentration of nitric oxide rapidly from 0.4 μM to 16.9 μM compared to the usual. However, when LPS-induced raw 264.7 cells were treated with a small fennel extract at a concentration (5, 25, 50, or 100 μg / mL), the concentrations of nitric oxide increased to 16.9 μM were 15.6, 12.3, and 8.0, respectively. , 3.0 μM was confirmed to decrease. This means that the fennel extract has an inhibitory effect on the inflammatory response induced by LPS in macrophages.

(3) Determination of the effect of small fennel extract on the inhibition of nitric oxide synthase (iNOS) expression

Raw 264.7 cells were planted in 6-well plates at a number of 1 × 10 ⁶ cells / mL and incubated for 4 hours. In each well of a 6-well plate, a small fennel extract was added at each concentration (5, 25, 50, or 100 μg / mL) and incubated in the incubator for 30 minutes. Thereafter, LPS was added to each well at a concentration of 100 ng / mL. The 6-well plates were incubated for a time that matched the conditions of the protein to be identified in the incubator. Each well was washed twice with 1 × PBS, and homogenized by adding pro-prep and reacting at 4 ° C. for 30 minutes. The cells were scraped using a scraper and placed in a 1.5-mL tube to repeat the vortex and sonication three times. After reacting in ice for 1 hour, the supernatant was taken by centrifugation at 12000 rpm and 4 ° C for 10 minutes. Protein quantification was quantified using the Bradford method. Protein lysate was mixed with 8 × SDS loading buffer, boiled for 5 minutes, and separated by electrophoresis through 10% polyacrylamide gel. The separated proteins were transferred to PVDF membrane, blotted and blocked in 5% skim milk solution. Thereafter, iNOS monoclonal antibody (1: 2000) was diluted in 5% skim milk solution and reacted overnight. After washing three times with TBS-T, horseradish peroxidase-anti rabbit IgG secondary antibody (1: 5000) was diluted in 5% skim milk solution and reacted at room temperature for 1 hour. After washing three times with TBS-T, using the enhanced chemiluminescence (ECL) detection reagent, the color reaction through the antigen-antibody reaction was confirmed by photosensitive to the X-ray film. The results are shown in FIG.

It was confirmed that the expression of iNOS protein was sharply increased in the control group treated with LPS alone. In addition, the small fennel extract was treated with each concentration (5, 25, 50, or 100 ㎍ / mL), it was confirmed that the concentration-dependent decrease.

Through this, the small fennel extract was found to reduce the concentration of nitric oxide by inhibiting the expression of iNOS protein in Raw 264.7 cells induced by LPS.

(4) Determination of the effect of small fennel extract on NF-κB activity

Raw 264.7 cells were planted in 6-well plates at 1 × 10 ⁶ cells / mL and incubated for 4 hours. Opti-MEM medium: lipofectamine 2000 reagent: DNA was mixed at a ratio of 100 mL: 1 mL: 2 mg, respectively, and reacted at room temperature for 20 minutes. The DNA used was co-transfected with pELAM and pCMV-luc. The 6-well plates were washed twice with serum free medium and the lipofectamine mixture was added to 1/5 of the culture medium and incubated in the incubator for 24 hours. In each well of a co-transfected 6-well plate, a small fennel extract was added at each concentration (5, 25, 50, or 100 μg / mL) and incubated in the incubator for 30 minutes. Thereafter, LPS was added to each well at a concentration of 100 ng / mL. It was incubated in incubator for 1 hour. NF-κB luciferase activity was measured using a Dual Luciferase Reporter Assay System. At this time, firefly luciferase activity (Firefly luciferase activity) was quantified by sea fancy Luciferase activity (Renilla luciferase activity). The results are shown in Fig.

In the control group treated with LPS alone, activation was increased by about five times compared to the group treated with nothing. In addition, the concentration of NF-κB was decreased depending on the concentration of small fennel extract, and it was significantly reduced to the level of the no-treated group in the experimental group treated with 50 or 100 ㎍ / mL. Activation has been shown to decrease. This suggests that the fennel extract prevents the degradation of IkB and directly inhibits NF-κB activity.

(5) measurement of cell viability

Raw 264.7 cells were planted in 96-well plates at 1 × 10 ⁶ cells / mL and incubated for 4 hours. In each well of a 96-well plate, a small fennel extract was added at each concentration (5, 25, 50, or 100 μg / mL) and incubated for 23 hours in an incubator. Each well of a 96-well plate was charged with an Ez-cytox kit at a dose of 1/10 and further incubated for 1 hour. Cytotoxicity was measured by absorbance at 450 nm wavelength using a microplate reader. At this time, toxicity was measured based on the cells that did not process anything. The results are shown in FIG.

As shown in FIG. 4, cell viability was not affected until 24 hours in the group treated with small fennel extract at 5, 25, 50 and 100 μg / mL and LPS.

Next, the present invention will be described in more detail as formulation examples.

Preparation Example 1

Small Fennel Extract 100mg

Appropriate sterile distilled water for injection

pH adjuster

The fennel extract is dissolved in distilled water for injection, adjusted to pH of about 7.6 with a pH adjuster, the total amount is 2 mL, and then filled into a 2 mL ampoule and sterilized to prepare an injection.

Preparation Example 2

Small Fennel Extract 10mg

Lactose 100mg

Starch 50mg

Magnesium stearate proper amount

The above components are mixed and tablets are prepared by tableting according to a conventional method for producing tablets.

Preparation Example 3

Small Fennel Extract 5mg

Lactose 100mg

Starch 93mg

Tackle 2mg

Magnesium stearate proper amount

The capsules are prepared by mixing the above components and filling gelatin capsules according to a conventional method for preparing capsules.

Preparation Example 4

Small Fennel Extract 100mg

20 g of sugar

20g per isomer

Lemon incense quantity

Add 100 mL of purified water

The above components are mixed according to a conventional method for preparing a liquid, and filled into 100 mL of brown bottle and sterilized to prepare a liquid.

Claims (10)

Sashimi ( Anethum) graveolens ) A composition for the prevention or treatment of inflammatory diseases comprising a flower extract as an active ingredient.
The method of claim 1,
The small fennel flower extract is water, a lower alcohol having 1 to 6 carbon atoms or a mixed solvent thereof, or an organic solvent fraction of the lower alcohol extract, a composition for preventing or treating inflammatory diseases.
The method of claim 1,
Inflammatory disease is a composition for the prevention or treatment of inflammatory diseases, such as rheumatoid arthritis or asthma.
The method of claim 1,
A composition for preventing or treating an inflammatory disease further comprising a pharmaceutically acceptable carrier.
The method of claim 1,
A composition for preventing or treating an inflammatory disease having a formulation of any one of a capsule, a liquid, an injection, a soft capsule, a granule, or a tablet.
Sashimi ( Anethum) graveolens ) A dietary supplement for the prevention or improvement of inflammatory diseases comprising flower extract as an active ingredient.
The method according to claim 6,
The small fennel flower extract is water, a lower alcohol having 1 to 6 carbon atoms or a mixed solvent thereof, or an organic solvent fraction of the lower alcohol extract.
The method according to claim 6,
Inflammatory diseases are health supplements for the prevention or amelioration of inflammatory diseases such as rheumatoid arthritis or asthma.
The method according to claim 6,
A dietary supplement for the prevention or amelioration of an inflammatory disease, characterized in that it is a food or food additive comprising an effective amount of a small fennel flower extract.
The method according to claim 6,
A health supplement for the prevention or amelioration of an inflammatory disease, characterized in that it is a beverage or a beverage additive comprising an effective amount of small fennel flower extract.

Images