KR20090056167A - Anti-inflammatory composition containing rubus coreanum extract as an effective factor - Google Patents

Abstract

A composition containing a Rubus coreanum extract for anti-inflammation is provided to suppress the activationof NF-Kb, cyclooxygenase-2, and chondroitinase and inhibit the inflammation. A pharmaceutical composition for anti-inflammation comprises a Rubus coreanus extract as an active ingredient. The Rubus coreanus extract is obtained through the hot water extraction or methanol extraction. A daily dose of the composition is 1-30 mg/kg. The composition is used in a form of tablet, pill, granule, capsule, suspension, emulsion, syrup, insoluble solvent, freeze-dried preparation or suppository. A fermented milk, beverage, or health functional food for anti-inflammation comprises the Rubus coreanus extract which is isolated with a hot water as an active ingredient.

Description

Anti-inflammatory composition containing Rubus coreanum extract as an effective factor

The present invention relates to a bokbunja extract having an anti-inflammatory effect and a composition containing the same as an active ingredient, and more particularly, the present invention relates to NF-κB, cyclooxygenase-2, chondroitinase ( It relates to a bokbunja extract having anti-inflammatory efficacy through the inhibition of the 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 any organic changes in cells or tissues. Local vascular, 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, while 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 as described above, the bokbunja extract 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 an anti-inflammatory effect to complete the present invention.

The present invention by inhibiting NF-κB, cyclooxygenase-2 (Cyclooxygenase-2), chondroitinase (chondrointinase) bokbunja extract having anti-inflammatory effect and an anti-inflammatory composition containing the same as an active ingredient, fermented milk, An object of the present invention is to provide a beverage and a dietary supplement.

In order to achieve the above object, the present invention is a bokbunja extract having anti-inflammatory effect by inhibiting NF-κB, cyclooxygenase-2, chondroitinase (chondrointinase) as an active ingredient It is characterized by providing an anti-inflammatory composition containing, fermented milk, beverages and health functional food.

Hereinafter, the present invention will be described in detail.

Bokbunja (Rubus coreanus) is a medicinal herb made from the raw fruit of the Rubus coreanus Miquel. Its shape is composed of several small lumps, forming a conical or pressed sphere. The outer side is yellowish green or light brown, and the end is round and round, and the center of the calyx is recessed. Bokbunja reinforces the function of the kidney (유 精), Mongjeong (夢 精), urea (遺尿), etc. are used to weaken eyesight, lighten the body and blacken the head. It also makes the skin soft and beautiful. Antioxidative and anti-Helicobacter pylori activity has been reported as a pharmacological action, but the anti-inflammatory mechanism and anti-inflammatory action are not known so far.

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

In the case of hot water extraction, the weight of the bokbunja sample was measured, and after crushing the sample, 10 times of distilled water was added and extracted at 110 ° C. for 3 hours. The freeze-dried bokbunja sample is 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 bokbunja sample, 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 and concentration in a distillation, after distilled water of 2 times the concentration of the concentrate was added and frozen, freeze-drying was performed, the weight of the freeze-dried Bokbunja sample was weighed, and the appropriate amount of DMSO was added to dissolve the methanol extract of Bokbunja. Store in freezer until use.

The anti-inflammatory pharmaceutical composition containing the bokbunja extract 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 bokbunja extract having an anti-inflammatory effect according to the present invention as an active ingredient can be formulated into various 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 may include at least one excipient (e.g., starch, sucrose, lactose and gelatin) in the extract of Bokbunja. It is mixed and 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 1-30 mg / kg body weight and may be administered once or divided into several 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.

Bokbunja extract of the present invention is a bokbunja used as an edible or medicinal raw material has little toxicity and side effects, so it is a preparation that can be used safely even for long-term use for the purpose of preventing or treating inflammation.

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

The anti-inflammatory fermented milk containing hot water extract of Bokbunja 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) In a culture solution, the juice concentrate, dietary fiber, glucose, oligosaccharides, calcium, vitamins, hot water extracts of bokbunja, etc. are prepared by mixing, stirring and homogenizing the syrup at a predetermined ratio to prepare fermented milk.

In addition, the anti-inflammatory drink containing the hot water extract of Bokbunja as an active ingredient, in addition to the hot water extract of Bokbunja as an active ingredient, and added to the calcium bottle, concentrate, liquid fructose, purified water, etc. After sterilization it is cooled to room temperature to prepare a beverage.

In addition, healthy functional foods for preventing inflammation containing hot water extract of Bokbunja as an active ingredient are nutritional supplements (vitamin B1, B2, B5, B6, E and acetate ester, nicotinic acid amide), oligosaccharide, 50 After mixing and adding% ethanol and purified water to form granules and drying them in a vacuum dryer, the granules are uniformly prepared by passing through 12 to 14 mesh and extruded by appropriate amounts to form tablets or powders or filled into hard capsules. To produce a hard capsule product.

As described above, the bokbunja extract of the present invention has an excellent anti-inflammatory effect by inhibiting NF-κB, cyclooxygenase-2, chondroitinase (chondrointinase) to treat inflammatory diseases And pharmaceutical compositions for the purpose of 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 Bokbunja Extract

Hydrothermal Extract of Bokbunja

Bokbunja was washed with water, air dried, weighed, and ground at 1 cm intervals. 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. 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 bokbunja.

The bokbunja hot water extract 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 Bokbunja

Bokbunja was washed with water, air dried, weighed, and ground at 1 cm intervals. 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 was added, frozen, and freeze-dried to prepare methanol extract of bokbunja.

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>

Pharmaceutical Compositions Using Bokbunja Extracts

The preparation example of the pharmaceutical composition containing the bokbunja extract of the present invention as an active ingredient will be described, but the present invention is not intended to be limited thereto, but is intended to be described in detail only.

Preparation of Capsules

100 mg of bokbunja 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 to prepare capsules.

Manufacture of powder

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

<Example 3>

Fermented Milk Production Using Bokbunja 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 the culture was incubated at 37 ° C. and cooled 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 oligosaccharide, 0.001-10% by weight of calcium, 0.0001-5% by weight of vitamins, and Bokbunja hydrothermal extract 0.01 After dissolving ~ 5% by weight, sterilized and cooled. 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 Bokbunja Hot Water Extract

After mixing 14% by weight of hot water extract of Bokbunja, 1.5% by weight of jujube extract and 7% by weight of sugar extract, liquid fructose and 7.4% by weight of purified fructose were added and mixed into the drink bottle and sterilized at 90-95 ° C. Cooled to produce a beverage.

<Example 5>

Health functional food manufacturing using bokbunja hot water extract

Nutritional supplements (vitamins B1, B2, B5, B6, E and acetic acid esters, nicotinic acid amide), oligosaccharides, and 50% ethanol in 0.01 to 30% by weight of bokbunja hydrothermal extract 5-10 based on 100% by weight of bokbunja hydrothermal extract Add to weight percent and mix 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 Extract of Bokbunja

The methanol extract sample of Bokbunja prepared in Example 1 was used to measure the inhibitory activity of NF-κB, which plays a major role 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 100 μg / ml of Bokbunja methanol extract sample to activate NF-κB. after 16 hours ^{Bright-Glo TM luciferase Assay System (} Promega社) using a kit of inducing luciferase reaction and in luminometer (luminometer, Biotek社) measuring the response value, and examined the inhibition of NF-κB.

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

<Test Example 2>

Determination of NF-κB Inhibitory Activity by Concentration of Hot Water Extract of Bokbunja

NF-κB inhibitory activity according to the concentration of Bokbunja 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, as the concentration of the bokbunja hydrothermal extract increases NF-κB inhibitory activity was increased and the IC ₅₀ of the bokbunja hydrothermal extract was about 40㎍ / ㎖.

<Test Example 3>

Of Bokbunja Hydrothermal Extract In vitro  Determination of COX-2 Enzyme Inhibitory Activity

The effects of COX-2 enzymes, which play an important role in the inflammatory response, were investigated using hot water extracts of bokbunja. COX-2 enzyme inhibitory activity was measured by the following calculation 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 and the bokbunja hydrothermal extract to be measured were mixed for each concentration 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 bokbunja hot water extract increased COX-2 enzyme inhibitory activity and the IC ₅₀ of the bokbunja hot water extract was found to be 25μg / ㎖.

<Test Example 4>

Measurement of COX-2 Promoter Inhibitory Activity of Bokbunja Hot-water Extract

The effects on the regulation of COX-2 promoter expression of bokbunja hydrothermal extract were investigated. The inhibitory activity of COX-2 promoter is luciferase when COX-2 is activated by 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 IL-1β to activate NF-κB and bokbunja hydrothermal extract samples of various concentrations, and again after 16 hours, the Bright-Glo ^TM Luciferase Assay System The kit was used to induce the reaction of luciferase, and the reaction value was measured by a luminometer (Biotek, Inc.) and the inhibition rate of the COX-2 promoter was investigated. The results are shown in FIG.

As can be seen in Figure 3, as the concentration of the bokbunja hot water extract increased the inhibition rate of the COX-2 promoter was found that the IC ₅₀ of the bokbunja hot water extract is 38μg / ㎖.

<Test Example 5>

Determination of Chondroitinase Enzyme Inhibitory Activity Using Hot Water Extract of Bokbunja

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). We investigated whether chondroitin activity was inhibited in vitro of bokbunja hydrothermal extract.

Chondroitinase activity was measured using absorbance. Specifically, 25 mU / ml chondroitinase (Sigma) was added to 50 mM Tris HCl buffer (60 mM Sodium acetate, pH 8.0) and bokbunja hot water extract was added for each concentration 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, as the concentration of the bokbunja hot water extract increased the activity inhibition rate of chondroitinase and the IC ₅₀ of the bokbunja hot water extract was found to be 11㎍ / ㎖.

<Test Example 6>

Cytotoxicity Test of Hot Water Extract of Bokbunja

MTT assay was carried out using Roche's Cell Proliferation KIT I (MTT) to examine the effects of Bokbunja hydrothermal extract on cytotoxicity and cell proliferation in Caco-2 cells, and Promega CellTiter-Glo ^ㄾ Luminescent The cell viability assay kit was used for the investigation. 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 was not confirmed when the concentration of 10 ~ 200㎍ / ㎖ hot water extract of bokbunja. Therefore, the cytotoxicity of the hydrothermal extract of Bokbunja was considered safe within the tested concentration range.

1 is a graph showing the results of measuring the NF-κB inhibitory activity of each concentration of hot water extract of bokbunja.

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

Figure 3 is a graph showing the results of measuring the inhibitory effect of COX-2 promoter activity by concentration of hot water extract of bokbunja.

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

Figure 5 is a graph showing the results of measuring the cytotoxicity by concentration of the hot water extract of bokbunja by MTT assay.

Figure 6 is a graph showing the results confirmed by the cell proliferation (Vill proliferation) and survival analysis (Viability Assay) of the concentration of hot water extract of bokbunja.

Claims (12)

Bokbunja extract with anti-inflammatory efficacy. The method of claim 1, The bokbunja extract is bokbunja extract having an anti-inflammatory effect, characterized in that it inhibits NF-κB. The method of claim 1, The bokbunja extract is bokbunja extract having an anti-inflammatory effect, characterized in that it inhibits cyclooxygenase-2 (Cyclooxygenase-2). The method of claim 1, The bokbunja extract is bokbunja extract having an anti-inflammatory effect, characterized in that it inhibits chondroitinase (chondrointinase). A pharmaceutical composition for anti-inflammatory, comprising the extract of claim 1 as an active ingredient. Claims 2 to 4 of any one of the anti-inflammatory pharmaceutical composition comprising the extract of Bokbunja as an active ingredient. Fermented milk for preventing inflammation, comprising hot water extract of Bokbunja of claim 1 as an active ingredient. An anti-inflammatory fermented milk, comprising hot water extract of Bokbunja of any one of claims 2 to 4 as an active ingredient. Inflammation-preventing beverages comprising hot water extract of Bokbunja of claim 1 as an active ingredient. Inflammation-preventing beverages containing hot water extract of Bokbunja of any one of Claims 2-4 as an active ingredient. Anti-inflammatory health functional food comprising a hot water extract of Bokbunja of claim 1 as an active ingredient. Claims 2 to 4 of the functional food for preventing inflammation, characterized in that it contains a hot water extract of bokbunja of any one of the claims as an active ingredient.

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