KR20090056170A - Anti-inflammatory composition containing geranium koreanum extract as an effective factor - Google Patents

Abstract

A composition containing Geranium nepalense sub sp. thunbergii extract for anti-inflammation is provided to prevent and treat inflammatory diseases through the inhibition of NF-kB, cyclooxygenase-2, and chondroitinase. A pharmateutical composition for anti-inflammation comprises Geranium nepalense sub sp. thunbergii extract having the anti-inflammatory effect suppressing the NF-kappaB, cyclooxygenase-2, and chondroitinase. The Geranium nepalense sub sp. thunbergii extract is obtained by washing, pulverizing, extracting in water at 110°C for three hours, filtering, compress-concentrating, and freeze-drying. The daily dose of the composition is 1-30 mg/kg, and administrated once or several times. A fermented milk, beverage, or health functional food for preventing the inflammation comprises the Geranium nepalense sub sp. thunbergii extract.

Description

Anti-inflammatory composition containing Geranium koreanum extract as an effective factor

The present invention relates to a rounded grass extract having an anti-inflammatory effect and a composition containing the same as an active ingredient, more specifically, the present invention NF-κB, cyclooxygenase-2 (Cyclooxygenase-2), chondroitinase It relates to a rounded grass extract having anti-inflammatory effect through the inhibition of activation of (chondroitinase) and an anti-inflammatory composition, fermented milk, beverages and health functional foods containing the same as an active ingredient.

Inflammatory reactions are biological defense reactions for repairing and repairing damage caused by invasion that causes certain organic changes in cells or tissues. Local blood vessels, various tissue cells of body fluids and immune cells work. Normally, the inflammatory response induced by external invading bacteria is a defense system for protecting the living body, whereas abnormally excessive inflammatory response is induced, and various diseases appear. These diseases are called inflammatory diseases. The inflammatory disease is a disease that threatens human life by amplifying and sustaining inflammation of various inflammatory mediators secreted from target cells activated by external stimuli, such as chronic inflammatory diseases in joints such as acute inflammation and rheumatoid arthritis. have.

The key inflammatory mediators that induce these inflammatory diseases are prostaglandins and hydroxy fatty acids, which are precursors of arachidone by cyclooxygenase (COX) and lipoxygenase. It is produced from arachidonic acid.

However, due to westernized diet, lack of exercise, and environmental pollution, various acute and chronic inflammatory diseases are gradually increasing in modern people. Antibiotics and anti-inflammatory drugs are used as treatments for various inflammatory diseases, and various side effects of drugs are gradually becoming a problem. In order to treat inflammation, various anti-inflammatory substances have been developed, and anti-inflammatory substances reported to date are substances that inhibit the biosynthesis of prostaglandins that are activated in damaged tissue cells and cells involved in inflammation in acute inflammation. These substances have a therapeutic effect on acute inflammation, but only alleviate the primary inflammation for chronic inflammation such as rheumatoid arthritis, and can not expect immunological therapeutic effects and also has various side effects including cardiovascular system.

Therefore, while studying the natural plant-derived plant extract that can be used safely without any problems such as various side effects and resistance, such as excellent effect on chronic inflammation, the extract of the rounded grass is NF-κB, cyclooxygenase-2 (Cyclooxygenase-2) , Hereinafter referred to as 'COX-2'), by inhibiting the activity of chondroitinase (chondroitinase) has been found to have anti-inflammatory efficacy to complete the present invention.

The present invention is an anti-inflammatory composition containing anti-inflammatory activity and anti-inflammatory composition containing the same as an active ingredient, fermented milk by inhibiting NF-κB, cyclooxygenase-2, chondroitinase (chondrointinase) It aims to provide beverages and nutraceuticals.

In order to achieve the above object, the present invention is effective in the extract of rounded grass having anti-inflammatory effect by inhibiting NF-κB, cyclooxygenase-2, chondroitinase (chondrointinase) and effective It is characterized by providing an anti-inflammatory composition, fermented milk, beverages and health functional foods containing as a component.

Hereinafter, the present invention will be described in detail.

Geranium nepalense sub sp. Thunbergii belongs to the rat handiwork family and is distributed in Gyeongsangnam-do, Gyeonggi-do, Gangwon-do, Hwanghae-do, Pyongan-namdo, Pyongan-bukto, and Hamgyongnam-do. The whole has a little hairs and the stem is straight and branches. The height is about 1m. The leaves are opposite, the leaves from the roots have long petioles, and the leaves from the stems have little or no petioles. The leaves are 3 ~ 5 pieces, deeply cracked, and the split pieces have pointed ends and large jagged teeth. Jaw leaves are broad oval, membranous. Flowers bloom pale pink in June-July and hang on inflorescences about 2cm in diameter. Petals are 5, egg-shaped, 5 calyx pieces. There are 10 stamens with hairs on the base of the operating table and 1 pistil. Fruits are capsules, hairy, about 3cm long, open in 5 minutes. The entire grass is used for medicinal purposes, but its anti-inflammatory mechanism and anti-inflammatory activity are not known.

Looking at the process of efficiently separating the extract of the round heterogeneous grass having an anti-inflammatory effect as follows.

In the case of hot water extraction, weigh the sample of round heterosoil, crush the sample, add 10 times distilled water, extract for 3 hours at 110 ° C, filter twice with Whatman filter paper, concentrate with a distillation, and freeze. After drying, freeze-dried rounded grass samples are weighed, dissolved in an appropriate amount of distilled water, filtered through a 0.2 μm filter and stored in a freezer until use.

In the case of methanol extraction, weigh the sample of round heterosoil, crush the sample, add methanol with a purity of 99.9% or more of 5 times the sample weight, and store it for 72 hours at room temperature to induce extraction. After filtration twice, concentrated with a distiller, added 2 times the volume of distilled water and freezing, lyophilizing, weighing the freeze-dried round grass samples, and adding the appropriate amount of DMSO to dissolve the round grass extract. Store in freezer until use.

The anti-inflammatory pharmaceutical composition containing the extract of rounded grass having an anti-inflammatory effect according to the present invention as an active ingredient may be administered through various routes including oral, transdermal, subcutaneous, intravenous or intramuscular. In addition, the anti-inflammatory pharmaceutical composition containing the extract of the round rim grass having an anti-inflammatory effect according to the present invention as an active ingredient can be formulated into a variety of formulations, when formulated, commonly used fillers, extenders, binders, wetting agents It may be formulated using diluents or excipients such as disintegrants and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, which solid preparations contain at least one excipient (e.g., starch, sucrose, lactose and gelatin) in the extract of the rounded grass The mixture is prepared. Lubricants may also be used in addition to simple excipients. Liquid preparations for oral administration include suspensions, solvents, emulsions, and syrups. In addition to the commonly used simple diluents, water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives, etc. May be included. Formulations for parenteral administration include sterile aqueous solutions, water-insoluble solvents, suspensions, emulsions, lyophilized formulations and suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. Glycerol and gelatin may be used as the base of the suppository.

For humans, a typical daily dose may range from 1 to 30 mg / kg body weight and may be administered once or in divided doses. However, the actual dosage should be determined in light of several relevant factors such as route of administration, age, sex and weight, and health status.

Rounded grass herb extract of the present invention is a preparation that can be used safely in long-term use for the purpose of preventing or treating inflammation because there is little toxicity and side effects as a raw round grass used as an edible or medicinal.

On the other hand, the present invention can be used as an anti-inflammatory fermented milk, beverages and health functional foods containing the hot water extract of the rounded grass obtained by the extraction method as an active ingredient.

The anti-inflammatory fermented milk containing the hot water extract of the rounded grass as an active ingredient is lactobacillus casei HY2782 { Lactobacillus casei HY2782; Deposited Organization: Korean spawn association, Accession No. KFCC-10813, Date of Deposit, December 7, 1993, Patent Registration No. 0125219 (Name of invention: Lactobacillus skazy HY2782 and product using it) Dissolve juice concentrates, dietary fiber, glucose, oligosaccharides, calcium, vitamins, hot water extracts of round grass, etc. in a culture solution mixed with a certain ratio, stirred, homogenized and then packaged in a container to prepare fermented milk.

In addition, the anti-inflammatory drink containing the hot water extract of the rounded grass as an active ingredient, in addition to the hot water extract of the rounded grass is included as an active ingredient, drink by adding and mixing calcium, kaoshigapi concentrate, liquid fructose, purified water, etc. The bottle is filled and sterilized, and then cooled to room temperature to prepare a beverage.

In addition, the health functional food for preventing inflammation containing the above-mentioned hot water extract of rounded grass is an active ingredient (vitamin B1, B2, B5, B6, E and acetic acid ester, nicotinic acid amide), oligosaccharide in the hot water extract of rounded grass , 50% ethanol and purified water were mixed and molded into granules, dried in a vacuum dryer, and then passed through 12 to 14 mesh to make granules uniformly, followed by extrusion molding to form tablets or powders or hard capsules. It is filled in to prepare a hard capsule product.

As described above, the rounded grass extract of the present invention has an excellent anti-inflammatory effect by inhibiting NF-κB, cyclooxygenase-2 (Cyclooxygenase-2), chondroitinase (chondrointinase) of inflammatory diseases It may be used as a pharmaceutical composition for the purpose of treatment and prevention.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are not intended to limit the scope of the present invention, it is possible for the ordinary change by those skilled in the art within the scope of the technical idea of the present invention.

<Example 1>

Separation and Purification of Round Grass Extract

Hydrothermal Extract of Rounded Grass

After purchasing the round heteroglossy, washed with water and air-dried, weighed and pulverized at intervals of 1cm in length. 1 kg of the pulverized sample was placed in an extraction bottle, and 10L of distilled water was added thereto, followed by extraction at 110 ° C. for 3 hours, followed by primary filtration with 60 mesh and secondary filtration with filter paper (Whatman No. 41). The filtrate by the filter paper was concentrated under reduced pressure using a vacuum rotary concentrator (Eyela NE, Japan), and then lyophilized to prepare a hot water extract of a rounded grass.

The natural product was weighed, dissolved in an appropriate amount of distilled water, filtered through a 0.2 μm filter, and stored in a freezer until use. 10 mg of the dried sample was dissolved in purified water at a concentration of 1 ml and used as an anti-inflammatory efficacy measurement material.

Methanol Extract of Round Grass

After purchasing the round heteroglossy, washed with water and air-dried, weighed and pulverized at intervals of 1cm in length. 1 kg of the pulverized sample was placed in an extraction bottle, 5 L of methanol having a purity of 99.9% or more was added thereto, followed by extraction for 72 hours at room temperature, followed by primary filtration with 60 mesh and secondary filtration with filter paper (Whatman No. 41). The filtrate by the filter paper was concentrated under reduced pressure using a vacuum rotary concentrator (Eyela NE, Japan), and then distilled water of twice the concentration of the concentrate was added, frozen, and freeze-dried to prepare a methanol extract of rounded grass.

10 mg of the dried sample was dissolved in DMSO at a concentration of 1 ml and used as an anti-inflammatory efficacy measurement material.

<Example 2>

Preparation of pharmaceutical composition using extract of round grass

It describes an example of the preparation of a pharmaceutical composition containing a round grass extract of the present invention as an active ingredient, but the present invention is intended to explain in detail only, not intended to limit it.

Preparation of Capsules

100 mg, round starch extract, 100 mg of corn starch, 100 mg of lactose, and 2 mg of magnesium stearate were thoroughly mixed and then filled into hard gelatin capsules according to a conventional method for preparing capsules.

Manufacture of powder

2 g of rounded grass herb extract samples and 1 g of lactose were thoroughly mixed, and then filled in an airtight cloth to prepare a powder according to a conventional powder preparation method.

<Example 3>

Fermented milk production using hot water extract

Crude milk was sterilized for 15 seconds at 72 ~ 75 ℃ to adjust the nonfat milk solid content to 8 to 20% by weight. The sterilized crude oil was cooled to 37 ° C., and then inoculated with Lactobacillus casei HY 2782 at a concentration of 10 ⁶ cfu / ml, and then cultured and cooled at 37 ° C. until the culture solution had a pH of 4-5. Syrup contains 0.1-50% by weight of juice concentrate, 0.1-20% by weight of dietary fiber, 0.5-30% by weight of glucose, 0.1-15% by weight of oligosaccharides, 0.001-10% by weight of calcium, 0.0001-5% by weight of vitamins, and hot water extract It was prepared by dissolving 0.01-5% by weight, sterilizing and cooling. The syrup and the culture solution thus prepared were mixed and stirred at a volume ratio of 1: 3, homogenized, and then packaged in a container to prepare fermented milk. The produced fermented milk showed good results in flavor, physical properties and overall taste.

<Example 4>

Beverage Preparation Using Round Coniferous Hot Water Extract

After mixing 14% by weight of hot water extract of rounded grass, 1.5% by weight of jujube extract and 7% by weight of Angelica Concentrate, liquid fructose 7.4% by weight, add purified water to it and mix it into a drink bottle and sterilize at 90 ~ 95 ℃. The beverage was prepared by cooling to room temperature.

<Example 5>

Preparation of Health Functional Food Using Rounded Algae Hot Water Extract

0.01 to 30% by weight of rounded grass hot water extract, nutritional supplements (vitamins B1, B2, B5, B6, E and acetate esters, nicotinic acid amide), oligosaccharides, and 50% ethanol. 5-10% by weight was added and mixed in a high speed mixer. 10% by weight of sterile purified water was added to the mixture, mixed, and molded into granules having a diameter of 1 to 2 mm. The molded granules were dried in a vacuum dryer at 40 ° C.-50 ° C., and then uniformly prepared granules were passed through 12 to 14 mesh. The granules prepared as described above may be extruded by appropriate amounts into tablets or powders or filled into hard capsules to produce hard capsule products.

<Test Example 1>

Determination of NF-κB Inhibitory Activity of Methanol Extracts from Rounded Grass

Methanol extract samples of the rounded grass prepared in Example 1 were used to determine the inhibitory activity of NF-κB, which plays a role as a major factor in the mechanism of inflammation. For this, Caco-2 cell (ATCC HTB-37), a human colon cell-derived cell line, and three NF-κB transcriptional regulators are present in succession, followed by luciferase gene as a reporter gene. 3κB, a plasmid vector designed to express luciferase upon activation of κB was used.

As a test method, transfection was performed using 2.5 μg of 3κB plasmid and 6.25 μl of lipofectamine 2000 (Invitrogen, Inc.) on Caco-2 cells grown about 50% in T25 flasks. After 6 hours, cells were collected and transferred to a 96 well plate, simultaneously treated with 5ng / ml of Interleukin-1β (IL-1β) and 100μg / ml of rounded methanol extract to activate NF-κB. After 16 hours, the luciferase reaction was induced using a Bright-Glo ^™ Luciferase Assay System (Promega) kit, the reaction value was measured by a luminometer (luminometer, Biotek), and the inhibition rate of NF-κB was investigated.

As a result, it was confirmed that the methanol extract of the rounded grass showed high NF-κB inhibitory activity of 91%.

<Test Example 2>

Determination of NF-κB Inhibitory Activity by Concentrations of Hot Water Extracts

NF-κB inhibitory activity according to the concentration of C. hydrothermal extract was investigated by luciferase reporter assay using Caco-2 cells and 3κB plasmid vector as in Test Example 1. The results are shown in FIG.

As can be seen in Figure 1, NF-κB inhibitory activity increases as the concentration of the hot water extract of the round genus grass was increased, and the IC ₅₀ of the round genus hot water extract was about 39.5 µg / ml.

<Test Example 3>

Of algae hydrothermal extract In vitro  Determination of COX-2 Enzyme Inhibitory Activity

The hydrothermal extracts of rounded grass were used to investigate the effects of COX-2 enzymes, which play an important role in the inflammatory response. COX-2 enzyme inhibitory activity was measured using absorbance. Specifically, 50 nM of COX-2 was added to 100 mM Tris-HCl buffer solution (pH 8.0) at a final concentration, and 0.5 μM of Heme was added thereto. The COX-2 solution was mixed with each round hot water extract concentration to be measured and maintained for 5 minutes. The reaction was started by adding 100 μM of arachidonic acid and 10 μM of TMPD ( N, N, N ', N'-tetramethyl-p-phenylenediamine). After 5 minutes, the absorbance was measured at 590 nm. The results are shown in FIG.

Inhibition rate calculation

        C1: Absorbance of the positive control (test group with COX-2)

        C2: Absorbance of negative control group (test group without COX-2)

        S1: absorbance of sample group (test group with both COX-2 and sample)

        S2: Absorbance of the sample control group (test group without sample without COX-2)

As can be seen in Figure 2, as the concentration of the hot water extracts of rounded grass was increased COX-2 enzyme inhibitory activity and the IC ₅₀ of the rounded water pooled hot water extract was found to be 32μg / ㎖.

<Test Example 4>

Determination of COX-2 Promoter Inhibition Activity of Hot Water Extract

The effect on the regulation of the expression of COX-2 promoter of Rhizome hot water extract was investigated. The inhibitory activity of COX-2 promoter is luciferase upon COX-2 activation due to the presence of luciferase gene as Caco-2 cell (ATCC HTB-37) and COX-2 promoter and reporter gene. Was measured using a plasmid designed to be expressed. As a test method, transfection was performed using 2.5 μg of plasmid and 6.25 μl of lipofectamine 2000 (Invitrogen, Inc.) on Caco-2 cells grown about 50% in T25 flasks. After 6 hours, cells were collected and transferred to a 96 well plate, simultaneously treated with 5ng / mL of IL-1β and 100μg / mL round water extract of hot water extract to activate NF-κB and again after 16 hours, Bright-Glo ^TM Luciferase Assay The system (Promega) kit was used to induce the reaction of luciferase, and the reaction value was measured with a luminometer (Biotek) and the inhibition rate of the COX-2 promoter was investigated. The results are shown in FIG.

As can be seen in FIG. 3, as the concentration of the rounded hot water extract increased, the inhibition rate of the COX-2 promoter increased and the IC ₅₀ of the rounded hot water extract was 50 μg / ml.

<Test Example 5>

Determination of Chondroitinase Enzyme Inhibitory Activity Using Hot Water Extracts from Rounded Grass

Glycosaminoglycan (GAG) is present in the mucous region of normal gastrointestinal tissues, and the composition of glycosaminoglycans is different in inflammatory colitis patients compared to normal people. Chondroitin and dermatin are one of the major constituents of glycosaminoglycans. Therefore, it is possible to prevent or alleviate colitis by inhibiting the activity of chondroitinase, a chondroitin degrading enzyme.

In addition, Soll et al. Confirmed the alleviation of this inflammation by artificially injecting chondroitin sulfate from joints of humans and animals from trauma such as inflammation (US Pat. No. 5,498,606). In this regard, we investigated the activity inhibition of chondroitinase in vitro of hot water extract.

Chondroitinase activity was measured using absorbance. Specifically, 25 mU / mL chondroitinase (Sigma) was added to 50 mM Tris HCl buffer solution (60 mM Sodium acetate, pH 8.0), and the rounded hot water extract was added at different concentrations and maintained at room temperature for 5 minutes. 0.3 mg / mL chondroitin sulfate A (Sigman Co., Ltd.) was added thereto, followed by reaction at room temperature for 30 minutes, and then the reaction solution was reacted with a dimethylmethylene blue (DMB) solution. The absorbance was measured at 525 nm. The results are shown in FIG.

As can be seen in Figure 4, the increase in the concentration of round water extract hot water extract increased the inhibitory activity of chondroitinase and the IC ₅₀ of the round water extract was 21 µg / ㎖.

<Test Example 6>

Cytotoxicity Test of Hot Water Extracts from Rounded Grass

Processing the round cranesbill hot water extract using a Cell Proliferation KIT I (MTT) of Roche社the effect of cytotoxicity and cell proliferation (cell proliferation) in Caco-2 cells, by performing the MTT assay was CellTiter-Glo ^ㄾ of Promega社Luminescent Cell Viability Assay kit was used. The test methods were in compliance with the test methods recommended by each manufacturer. The results are shown in FIGS. 5 and 6.

As can be seen in Figures 5 and 6, significant toxicity could not be confirmed when the concentration of 10 to 200 ㎍ / ㎖ of hot water extract of the round zygophyte. Therefore, the cytotoxicity of the hydrothermal extracts of the round grass was judged to be safe within the tested concentration range.

1 is a graph showing the results of measuring the NF-κB inhibitory activity according to the concentration of the hot water extract of the round haricot grass.

Figure 2 is a graph showing the results of measuring the COX-2 inhibitory effect of each concentration of the hot water extract of the round haricot grass.

Figure 3 is a graph showing the results of measuring the inhibitory effect of COX-2 promoter activity according to the concentration of the hot water extract of the rounded grass.

Figure 4 is a graph showing the results of measuring the chondroitinase (chondroitinase) inhibitory effect of the concentration of the hot water extract of the round toothed grass.

Figure 5 is a graph showing the results measured by MTT assay for cytotoxicity by concentration of hot water extracts of round heteroglossy.

Figure 6 is a graph showing the results confirmed by the cell proliferation and viability assay (Viability Assay) according to the concentration of the hot water extract of the round toothed grass.

Claims (12)

Algae extract with anti-inflammatory effect. The method of claim 1, The round grass extract is a round grass extract having anti-inflammatory effect, characterized in that it inhibits NF-κB. The method of claim 1, The round grass extract is a round grass extract having anti-inflammatory effect, characterized in that it inhibits cyclooxygenase-2 (Cyclooxygenase-2). The method of claim 1, The round grass extract is a round grass extract with anti-inflammatory effect, characterized in that it inhibits chondroitinase (chondrointinase). A pharmaceutical composition for anti-inflammatory, characterized in that it comprises the extract of rounded grass of claim 1 as an active ingredient. The pharmaceutical composition for anti-inflammatory, characterized in that it comprises as an active ingredient the extract of the round myrtle of any one of claims 2 to 4. Anti-inflammatory fermented milk, characterized in that it contains a hot water extract of the rounded grass of claim 1 as an active ingredient. Anti-inflammatory fermented milk, characterized in that it contains a hot water extract of the rounded grass of any one of claims 2 to 4 as an active ingredient. Inflammation-preventing drink, characterized in that it contains hot water extract of the rounded grass of claim 1 as an active ingredient. Inflammation-preventing drink, characterized in that it contains a hot water extract of the rounded grass of any one of claims 2 to 4 as an active ingredient. Anti-inflammatory health functional food, characterized in that it contains a hot water extract of the rounded grass of claim 1 as an active ingredient. Claims 2 to 4 of the health functional food for preventing inflammation, characterized in that it contains a hot water extract of the round haricot according to any one of claims.

Images