KR100547560B1 - Pharmaceutical composition comprising the extract of Kalopanax pictus, Pueraria thunbergiana and Rhus verniciflua having anti-inflammatory activity. - Google Patents

Abstract

The present invention relates to a composition having anti-inflammatory effect, containing thawed bark, browned and lacquer wood extract or a mixture thereof, wherein the extract of the present invention (hereinafter referred to as KPR-2) inhibits NF-κB activity and thus LPS Since it exhibits an excellent anti-inflammatory effect by inhibiting the expression of induced iNOS and COX-2 genes, it can be used as a medicine and health functional food for the prevention and treatment of various inflammation-related diseases.

Anti-inflammatory, inflammatory disease, thawing blood, browning, lacquer

Description

Pharmaceutical composition comprising the extract of Kalopanax pictus, Pueraria thunbergiana and Rhus verniciflua having anti-inflammatory activity.}

1 is a diagram showing the effect of KPR-2 on the production of LPS-induced PGE2 in RAW 264.7 cells,

2A and 2B show the effect of KPR-2 on LPS-induced TNF-α reduction in RAW 264.7 cells, and FIG. 2A was treated with LPS alone and LPS alone at various concentrations of KPR-2 samples. Figure 2b shows the effect of the time, Figure 2b is a diagram showing the effect of KPR-2 through RT-PCR analysis,

3A and 3B show the effects of KPR-2 on LPS-induced iNOS and COX-2 protein and mRNA expression in RAW 264.7 cells, FIG. 3A shows KPR-2 at different concentrations with LPS alone and LPS. Figure 3 shows the effect when the sample is processed together, Figure 3b is a diagram showing the effect of KPR-2 through RT-PCR analysis,

4 is a diagram showing the degree of inhibition of NF-κB DNA binding by KPR-2,

5A and 5B are diagrams showing Western blot analysis for IκB degradation. FIG. 5A is a result obtained by treating RAW 264.7 macrophages with 1 μg / ml LPS and 30 μg / ml KPR-2. FIG. 5b. Shows results when 10, 30 and 60 µg / ml KPR-2 samples were treated.

The present invention relates to a thawing bark, browned and lacquer extract or a mixed extract thereof (KPR-2) having an anti-inflammatory activity and a composition containing the same, and to a pharmaceutical composition and health functional food for preventing and treating various inflammation-related diseases.

Inflammatory reactions are associated with various mediators and immune cells of local blood vessels and body fluids when infected with tissues (cells) or external infectious agents (bacteria, fungi, viruses, and various types of allergens). It has a series of complex physiological reactions, including substance secretion, fluid infiltration, cell migration, and tissue destruction, as well as external symptoms such as erythema, edema, fever, and pain. In normal cases, the inflammatory response removes external infectious agents and regenerates damaged tissues to restore the function of life.However, if the inflammatory response is excessive or persistent due to the absence of antigens or internal substances, the mucosal damage is promoted. Some lead to diseases such as cancer.

Various biochemical phenomena are involved as a cause of inflammation in the living body, and in particular, nitric oxide synthase (NOS) and prostaglandin, enzymes that generate nitric oxide (NO) Enzymes associated with biosynthesis are known to play an important role in mediating the inflammatory response. Therefore, NOS, an enzyme that produces NO from L-Arginine, or COX (cyclooxygenase), an enzyme involved in synthesizing prostaglandins from arachidonic acid, is a major target for blocking inflammation. have.

According to recent research, there are several types of NOS, such as bNOS (brain NOS) in the brain, neuronal NOS in the nervous system, and endothelial NOS in the vascular system. It is expressed at a level, and a small amount of NO produced by them plays an important role in maintaining normal homeostasis, such as inducing neurotransmission and vasodilation, whereas iNOS induced by various cytokines or external stimulants NO, which is rapidly overproduced by induced NOS, is known to cause cytotoxicity and various inflammatory reactions, and chronic inflammation is associated with increased iNOS activity (Miller et al ., Mediators of inflammation , 4, pp 387-396, 1995; Appleton L. et al ., Adv . Pharmacol . , 35, pp27-28, 1996).

In addition, there are two kinds of isoforms of cyclooxygenases. Cyclooxygenase-1 (COX-1) is always present in the cell, and is used to synthesize prostaglandins (PGs) necessary for cytoprotective action. In contrast, COX-2 is known to play an important role in causing an inflammatory response due to a rapid increase in cells during the inflammatory response (Weisz A., Biochem . J. , 316, pp209-215, 1996). Transcriptional inflammatory factors, including iNOS and COX-2, which enhance the degree of NO and PGs, are chronic diseases including various sclerosis, parkinson's disease, Alzheimer's disease and colon cancer. Related to the etiology of.

NF-κB is a nuclear protein of the Rel gene family. In the cytoplasm, NF-κB binds to I-κB and exists in an inactive form. However, NF-κB is reactive oxygen, TNF-α (tumor necrosis factor-α), I-κB kinase is activated by various stimuli such as chemokines and lipopolysaccharide (LPS) such as IL-1 (Interleukin-1), and then I-κB is released through phosphorylation. . NF-κB, composed of p50 and p65 heterodimers, is known to promote gene expression that, after being activated, migrates to the nucleus to induce an inflammatory response (Oh, GT et al ., Atherosclerosis , 159 (1)). , pp 17-26, 2001; Epstein, FH et al ., The New England Journal of Medicine , 336 (15), pp 1066-1071, 1997; Zhang WJ et al ., FASEB J , 15 (130), pp2423-2432, 2001; Denk, A. et al ., J. Biol . Chem . , 276 (30), pp28451-28458, 2001; Sahnoun Z. et al ., Physiology , 53 (4), pp315-339, 1998; Lindner V ., Pathobiology, 66 (6) , pp311-320, 1998; Landry, DB et al, Am J. Pathol, 151 (4), pp1085-1095, 1997;..... Gerritsen, ME et al, Am J . Pathol., 147 (2) , pp278-292, 1995).

Multifunctional cytokines such as TNF-α are not only expressed in normal tissues but also increased in the course of lesions, and play an important role in skin inflammation, particularly during cancer promotion. TNF-α has been reported to be associated with inflammatory skin diseases in humans (Verhoef, J. et al., J. Antimicrob . Chemother ., 35, 1996). The treatment of TNF-α with various inflammatory diseases and allergic symptoms was alleviated. TNF-α also acts as an endogenous cancer promoter by activating neutrophils and increasing hydrogen peroxide production. Therefore, proinflammatory molecules may be inhibited by inhibiting the expression of the cytokine TNF-α, which plays a pivotal role in the inflammatory stage closely related to the oncogenic stage, or by inhibiting the production of PGE2 due to COX-2 activity inhibition. There is a good chance that it will be possible to control the course of the lesion that is accompanied by an increase. When treated with antibiotics under bacterial infection, LPS released from the extracellular membrane while killing bacteria causes endotoxin shock and, when severely destroyed by this process, does not neutralize the LPS that continues to be produced (Dunn, DL, Surg Clin North Am, 74 , 1994;...... Giroir, BP, Crit Care Med, 21, 1993; Yamaguchi, Y. et al., J. Reticuloendothel. Soc ., 409, 1982). Therefore, drug treatment may be effective by reducing the action of LPS. Reduction of inflammation due to the use of nonsteroidal anti-inflammatory drugs (NSAIDs) to date has often resulted in relief of pain for a considerable period of time, and most of the non-narcotic analgesics (such as aspirin) are also anti- It has an inflammatory effect and has been used properly for the treatment of acute and chronic inflammatory diseases. However, due to side effects, it is difficult to use in the long term, and as a result, the development of new or improved therapeutics is essential and urgent, because the seriousness of the side effects is too great for the present treatment. In connection with the problem of overcoming these side effects, efforts have recently been made to find the active ingredient in natural products that are used in the private sector.

Kalopanax pictus THUNB of the present invention is a bark of chyme and related plants, and is a deciduous tree of the Araliaceae growing in the mountains of Korea. Bark and leaves of Yin tree contain 13 to 30% of tannins, bark and heart material contains polyacetylene compounds, and stem and leaf reactions of cardiac glycosides and anthra glycosides Appears. In addition, flavonoid glycosides, coumarin glycosides and small amounts of alkaloids, essential oils, saponins, and the like are contained. Saponins are triterpenoid-based saponins and aglycons are headagenin. Haedongpi is used to control the wind in Chinese medicine and summarizes neuralgia and rheumatism, and it is also used for low back pain.It is used in the private sector to treat neuralgia and low back pain by drinking tea and liquor with boiled water. Steaming affected areas with boiled bark and root bark is known to have an effect (Jeong Bo-seop and Shin Min-kyo, Hyangjedae, Yeonglimsa, p438, 1998).

Galhwa (葛花, Pueraria thunbergiana ) refers to the flower of the 칡, a medicine that relieves the poison and removes the intestinal poison. It is effective in fever, ill feeling, loss of appetite and hemostasis caused by resident wine (Jebo Bo-seop and Shin Mingyo, Hyangjeomsa, Yeonglimsa, pp704-705, 1998).

Sumac ( Rhus verniciflua STOKES) comes from the word 'paint', which is called Guilchi (乾 漆). Lacquer is very effective in treating various cancers and incurable diseases. Since lacquer is the best preservative and insecticide, it is very good to use for the treatment of cancer because the bacterium extinguished by the lacquer poison cannot be revived and the neutralized lacquer poison does not destroy the pigment of the human body. Lacquer is a digestive agent in the stomach, a blood pill in the liver to control inflammation, a blue blood agent in the heart to control all heart diseases, and a pesticide in the lungs to destroy tuberculosis bacteria. Yi Su Ya (利 水 藥) to control the disease of the five intestines. It is also effective in neuralgia, arthritis and skin diseases of the whole body (Bo Bo-seop and Shin Min-kyo, Hyang-dae metabolism, Younglimsa, pp382-383, 1998).

Herbal medicines classified as herbal decoctions and applied to diabetes have been reported to mainly inhibit NO, PGE2 and TNF-α produced by lipopolysacchride (LPS) by macrophage cells (Kim, YK. Et al. , Biological and Pharmaceutica Bulletin, 25, pp472-476, 2002; Jung HJ. Et al., Planta Medica ., 69, pp 610-616, 2003). Among them, the present inventors reported that the active ingredients of thawed skin, browning, and lacquer wood were used for inhibiting mutations, inhibiting lipid peroxidation, anti-inflammatory effect, and anti-diabetic effect. It has been reported to be highly related to the inhibitory effect of Park KY. Et al., Arch. Pharm . Res. , 25, pp320-324, 2002; Choi, JW. Et al, Journal of Ethnopharmacology , pp199-204, 2002 ; Choi JW, Yoon BJ. Et al., Nutrsceuticals and Food , 7, pp347-352, 2002; Shin, KH et al., Planta Med ., 65, pp 776-777, 1999).

Already, the present inventors have already found that kalopanaxsaponin A, liriodendrin and syringin and cytorigenin and kaikasaponin III of kaepanaxsaponin III and lacquer wood (sulfuretin) reported various pharmacological actions with respect to aging. The present inventors observed that the effects of these fractions were comparable to those of the active substance, and that there was a combination of herbal medicines (or mixed herbs) in the herbal medicines, assuming that the fractions or combinations of the herbal medicines would exhibit synergistic effects. The present invention was completed by confirming that it can be developed as a drug having no superior activity and no toxicity.

An object of the present invention is to provide a pharmaceutical composition for the treatment and prevention of inflammation containing thaw skin, browning and lacquer water or ethyl acetate soluble extract having an anti-inflammatory effect as an active ingredient.

In order to achieve the above object, the present invention provides a thaw skin, browning, lacquer extract or a mixed extract thereof having an anti-inflammatory effect.

The extract includes an extract extracted from a polar solvent such as C1 to C4 lower alcohols such as methanol, ethanol and butanol, a nonpolar solvent such as hexane, ethyl acetate, acetone, chloroform, methylene chloride or a mixed solvent thereof.

The present invention provides a pharmaceutical composition for the treatment and prevention of inflammation, containing thaw skin, browning and lacquer water or ethyl acetate soluble extract as an active ingredient.

 Hereinafter, the present invention will be described in detail.

Specifically, the thawed bark, browned and lacquer extract of the present invention is purchased by drying the thawed bark, browned and lacquer, and then pulverized and powdered, and then thawed bark, browned and lacquered each or about 2 to 20 times the weight of the mixed sample of the same weight. A polar solvent of up to the volume of water and C1 to C4 lower alcohols such as methanol, ethanol, butanol and the like, or a mixed solvent having a mixing ratio of about 1: 0.1 to 1:10, preferably 20 to 50 by adding water. After extraction using an extraction method such as hot water extraction, cold needle extraction, reflux cooling extraction or ultrasonic extraction for about 10 hours to 48 days, preferably 3 hours to 10 hours at ℃, it is filtered, concentrated and It can be dried to obtain the extract or mixed extract of thawed bark, browned and lacquer.

In addition, the non-polar solvent soluble extract of the present invention, after suspending the crude extract in distilled water, the suspension is about 1 to 100 times, preferably about 1 to 5 times the volume of non-polar solvents such as hexane, ethyl acetate, chloroform It can be obtained by extracting and separating the non-polar solvent soluble layer 1 to 10 times, preferably 2 to 5 times, and may be further subjected to a conventional fractionation process (Harborne JB Phytochemical methods: A guide to modern techniques of plant analysis, 3rd Ed., p6-7, 1998).

More preferred thaw, brown and lacquer soluble extracts of mixed or non-polar solvents can be prepared by suspending each of the above-mentioned thaw, brown and lacquer or mixed crude extracts in distilled water, and then about 1 to 100 times the suspension, preferably about 1 to 100 Non-polar solvents such as 5 times hexane, ethyl acetate, chloroform, and more preferably chloroform and ethyl acetate may be sequentially added to extract and separate the non-polar solvent soluble layer two to five times.

The present invention provides a pharmaceutical composition for the treatment and prevention of inflammation-related diseases containing the extracts of thawed bark, browned and lacquer tree obtained by the above method or a mixed extract thereof as an active ingredient.

The present inventors have tested the anti-inflammatory effects of the extracts or mixed extracts of the thawed bark, browned and lacquer trees obtained by the above preparation method, and the extracts of the present invention inhibited NF-κB activity, thereby expressing LPS-induced iNOS and COX-2 genes. It was confirmed that exhibited excellent anti-inflammatory activity by inhibiting.

In addition, thawed bark, brown flowers and lacquer trees have been used for a long time as a herbal medicine and edible extracts of the present invention extracted from them also have no problems such as toxicity and side effects.

The pharmaceutical composition for preventing and treating inflammation-related diseases of the present invention comprises 0.1 to 50% by weight of the extract based on the total weight of the composition.

Pharmaceutical compositions comprising the extract of the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions.

The pharmaceutical dosage forms of the extracts of the present invention may be used in the form of their pharmaceutically acceptable salts, and may be used alone or in combination with other pharmaceutically active compounds as well as in a suitable collection.

The pharmaceutical composition comprising the extract according to the present invention may be in the form of powder, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories, and sterile injectable solutions, respectively, according to a conventional method. Can be formulated and used. Carriers, excipients and diluents that may be included in the composition comprising the extract include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate , Cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and the solid preparations may include at least one excipient such as starch, calcium carbonate, sucrose in the extract. ) Or lactose, gelatin and the like are mixed. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

Preferred dosages of the extracts of the present invention vary depending on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration, and may be appropriately selected by those skilled in the art. However, for the desired effect, the extract of the present invention is preferably administered at 0.0001 to 100 mg / kg, preferably at 0.001 to 100 mg / kg. Administration may be administered once a day or may be divided several times. The dosage does not limit the scope of the invention in any aspect.

The extract of the present invention can be administered to mammals such as mice, mice, livestock, humans, etc. by various routes. All modes of administration can be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection.

The present invention provides a health functional food comprising the extract and a food acceptable food supplement additive exhibiting a prophylactic effect of inflammation-related diseases. Examples of the foods to which the extracts of thawed bark, brown flower and lacquer tree or mixed extracts thereof can be added include various foods, beverages, gums, teas, vitamin complexes, and health functional foods.

It may also be added to foods or beverages for the purpose of preventing the inflammation-related diseases. At this time, the amount of the extract in the food or beverage may be added in 0.01 to 15% by weight of the total food weight, the health beverage composition may be added in a ratio of 0.02 to 5 g, preferably 0.3 to 1g based on 100 ml. have.

The health functional beverage composition of the present invention is not particularly limited to other ingredients except for containing the extract as an essential ingredient in the indicated proportions, and may contain various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And conventional sugars such as polysaccharides such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (tauumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of natural carbohydrates is generally about 1-20 g, preferably about 5-12 g per 100 ml of the composition of the present invention.

In addition to the above, the extract of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring and neutralizing agents (such as cheese and chocolate), pectic acid and salts thereof, alginic acid and its Salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. In addition, the extracts of the present invention may contain flesh for the production of natural fruit juices and fruit juice beverages and vegetable beverages. These components can be used independently or in combination. The proportion of such additives is not so critical but is usually selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the extract of the present invention.

Below, The invention is illustrated in detail by the following examples and experimental examples.

However, the following Examples and Experimental Examples are merely illustrative of the present invention, and the content of the present invention is not limited by the following Examples and Experimental Examples.

Example  One. Thawed blood Water extract  Manufacture (K-1)

After thawing blood was purchased from Cheonil Pharmaceutical Co., Ltd., Wonju, and dried, ground to fine grinding machine, 4 liters of water was added to 1 kg of thawed blood fine powder sample, followed by reflux extraction at room temperature for 5 hours, and the supernatant was recovered by vacuum filtration. This process was repeated three times to collect the supernatant, and concentrated under reduced pressure to obtain 112 g of thawed blood extract, named K-1.

Example  2. Gall painting Water extract  Manufacture (P-1)

Purified galvanized from Cheonil Pharmaceutical Co., Ltd., Wonju-si, dried, milled with a crusher, and then finely ground, 4 kg of water was added to 1 kg of the ground fine powder, and then refluxed at room temperature for 5 hours, and the supernatant was recovered by vacuum filtration. This process was repeated three times to collect the supernatant, and then concentrated under reduced pressure to obtain 276 g of a brown water extract, named P-1.

Example  3. sumac Water extract  Manufacture (R-1)

Purified lacquer wood in Wonju-si traditional market, then dried and ground to fine grinding, milled with a crusher, 4 liters of water was added to 1 kg of lacquer fine powder sample, refluxed at room temperature for 5 hours, and the supernatant was recovered by vacuum filtration. . This process was repeated three times to collect the supernatant, and then concentrated under reduced pressure to obtain 27 g of lacquer extract, named R-1.

Example  4. Thawed blood , Gall painting  And sumac blend Water extract  Produce( KPR -One)

After drying the thawed bark, brown and lacquer trees purchased from Cheonil Pharmaceutical Co., Ltd. and Wonju City, respectively, grind them with a mill and grind them. After refluxing for 5 hours at room temperature, the supernatant was recovered by vacuum filtration. This process was repeated three times to collect the supernatant, and then concentrated under reduced pressure to obtain 125 g of thawed bark, browned and lacquered mixed water extract, which was named KPR-1.

Example  5. Thawed blood , Gall painting  And ethyl acetate of lacquer tree extract and mixed extract Fraction  Produce( KPR -2)

Each extract of thawed bark (K-1), browning (P-1) and lacquer tree (R-1) and mixed extract (KPR-1) obtained in Examples 1 to 4 were separately suspended in 1 L of distilled water, and then they were suspended in 1 L of chloroform. After fractionation 3 times, the remaining water layer was repeatedly fractionated three times with 1 L of ethyl acetate, and concentrated and vacuum-dried to obtain ethyl acetate fraction of each extract and mixed extract of thawed bark, lacquer and lacquer tree, and each of K-2 (4 g). ), P-2 (9 g), R-1 (16 g) and KPR-2 (6 g).

Experimental Example  1. Cell Culture and Reagents

 1-1. Cell culture

RAW 264.7 cells, a Murine macrophage cell line, were cultured in 37 ° C, 5% CO2 incubator using RPMI medium containing 100 units / ml penicillin-streptomycin and 10% fetal bovine serum (FBS) It was. After dispensing RAW 264.7 cells into 24 well plates at a concentration of 2 × 105 / ml, 10 μl of sample solutions of various concentrations (10, 30 and 60 μg / ml) were added the next day, and then 1 μg / ml LPS after 30 minutes. Was treated and incubated for 24 hours.

 1-2. reagent

RPMI medium, FBS and penicillin-streptomycin were purchased from Life Technologies Inc. (Grand Island, NY), and used for MTT reagent, dimethyl sulfoxide (DMSO), sulfanilamide, aprotinin, Leupetin, phenylmethylsulfonylfluride, dithiothreitol, NG-mono-methyl-arginine (NMA) and E. coli lipoplysaccaride (LPS) Was purchased from Sigma Chemicals, Inc. (MO, USA), and COX-2, iNOS, NF-κB (p65), anti-IκB-α single cell antibodies and peroxidase conjugated secondary antibodies were santa claus biotechnologies. It was purchased from the company (CA, USA). Prostarlandin E2 measurement kit was purchased from R & D Systems (MN, USA).

Experimental Example  2. Measurement of NO production inhibition

The amount of NO generated from the macrophage line was measured in the form of NO2- present in the cell culture using Greases reagent. Ie, 100 μl of cell culture supernatant and 100 μl of grease reagent (1% (w / v) sulfanylamide and 0.1% (w / v) naphthylethylenediamine-HCl) in 5% phosphoric acid solution, After reacting for minutes, the absorbance was measured at 550 nm. As a positive control, L-NMMA (IC 50 35.94 μM), known as an iNOS inhibitor, was used by substrate competition with L-arginine.

As a result, the most obvious inhibition of nitrite formation was shown in KPR-2, which was much better than KPR-1. Small amounts of nitrite (5.93 ± 0.9 μM) were produced in cells not treated with LPS.

NO MTT KPR-1 34.15 1184.65 KPR-2 5.11 476.48 R-1 22.00 346.52 R-2 16.68 153.61 K-1 43.24 401.87 K-2 10.95 317.59 P-1 55.76 622.36 P-2 26.10 152.95

Experimental Example  3. TNF -α and PGE2  Formation inhibition measurement

Cell cultures were taken and quantified respectively according to the instructions of the R & D kit. Preparation of RNA and RT-PCR RNA cells were allowed to divide into 5 × 10 6 cells in a 100 mm cell culture dish and then stabilized overnight. After treating the cells with the sample, 30 minutes later, the cells were treated with LPS, and after 24 hours, the cells were collected, washed with PBS, and 1 ml of easy blue (Intron) was added thereto, followed by stirring at room temperature. 200 μl of chloroform was added thereto, stirred again, and centrifuged at 13,000 rpm and 4 ° C. for 10 minutes. Then, isopropanol was added to 400 μl of the supernatant, followed by centrifugation to obtain RNA pellets. MuLV reverse transcriptase and 0.5 mM of 1 mM dNTP were added to the RNA obtained to prepare cDNA. These include primers from iNOS (sense 5′-CAACCAGTATTATGGCTCCT-3 ′, antisense 5′-GTGACAGCCCGGTCTTTCCA-3 ′) and COX-2 (sense 5′-CCGTGGTAATGTATGAGCA, antisense 5′-CTCGCTTCTGATCTGTCTT-3 ′) and GAPDH primers '-TCCCTCAAGATT-GTCAGCAA-3' and antisense 5'-AGATCCACAACGGATACATT-3 ') were added and amplified using a gene cycler (thermal cycler). At this time, iNOS was reacted for 1 minute at 95 ° C, 1 minute at 60 ° C, 1 minute 30 seconds at 72 ° C, and COX-2 for 1 minute at 94 ° C, 1 minute at 60 ° C, and 1 minute at 72 ° C. I was. The resulting RNA was electrophoresed on 2.5% agarose gel and confirmed by UV detector.

As a result, PGE2 formed by LPS treatment showed a significant concentration-dependent decrease in KPR-2 at 10, 30 and 60 μg / ml (see FIG. 1), and also significant in NS398 (10 μM) used as a reference drug. A decrease in PGE2 production was confirmed. In addition, it was confirmed that significantly reduced concentration-dependent TNF-α and mRNA expression at 10, 30 and 60 μg / ml KPR-2 (see FIG. 2).

Experimental Example  4. with COX-2 iNOS  Protein and mRNA  Expression inhibition

In order to investigate the correlation between the inhibition of inflammatory factors (NO and PGE2) by KPR-2 and the expression of iNOS and COX-2 proteins, Western blot and RT-PCR were used to investigate the protein expression.

Western blot test was performed by washing the cells treated with essential oils and the control group with PBS, followed by lysis buffer (50 mM HEPES pH 7.0, 250 mM NaCl, 5 mM EDTA, 0.15 Nonidet P-40, 1 mM phenylmethylsulfonyl flouride, 0.5 mM dithiothretol, Proteins were extracted with 5 mM NaF and 0.5 mM Na orthovanadate), followed by centrifugation to obtain supernatant and debris from cells. The supernatant was quantified with a bio-red reagent to take 50 μg of protein. The extracted protein was electrophoresed on a 10% SDS-polyarylamide gel, and then the protein of the gel was blotted onto a nitrocellulose membrane. After blocking overnight with 5% skim milk, iNOS and COX-2 antibodies were incubated at room temperature for 3 hours at a ratio of 1: 500, and then washed twice with TTBS every 15 minutes. It was. Rabbit and goat secondary antibodies diluted at a ratio of 1: 1000 were incubated at room temperature for 1 hour, washed again with TTBS for 15 minutes, and then developed by chemiluminescence. It was.

As a result, LPS significantly increased iNOS protein, KPR-2 inhibited iNOS concentration-dependently, and completely reduced protein expression at 60 μg / ml (see FIG. 3). In addition, the decrease of iNOS mRNA amount in RT-PCR analysis showed a similar tendency to protein level, and similar to iNOS, KPR-2 decreased COX-2 protein and mRNA induced by LPS. 4). Based on the above experimental results, it can be seen that the decrease of nitrite and PGE2 by KPR-2 is due to the regulation of protein expression. In contrast, 100 μM of NMMA (substrate analogue of iNOS enzyme) and 100 μM of SB203580 (specific inhibitor of COX-2) inhibited 55.0% and 61.98% of iNOS and PGE2 enzyme activities, respectively.

Experimental Example  5. KPR -2 LPS  Judo NF Determination of -κB Activity

RAW 264.7 macrophages were dispensed into 100 mm cell culture dishes at a concentration of 5 × 10 5 cells / ml. Cells treated with various concentrations (10, 30 and 60 μg / ml) of sample solution were activated with LPS for 1 hour, washed once with PBS, collected in 1 ml of cold PBS, and centrifuged. Collected cells were dissolved in hypotonic buffer (10 mM HEPES, pH 7.9, 1.5 mM MgCl 2, 10 mM KCl, 0.2 mM PMSF, 0.5 mM dithiothritol (DTT), 10 μg / ml aprotonin), and then on ice for 15 minutes. It was left. 0.1% Nonidet P-40 was added and vigorously stirred for 10 seconds to lyse the cells. The nucleus was allowed to settle by centrifugation at 12,000 g for 1 min at 4 ° C., which was then again concentrated in high salt buffer (20 mM HEPE, pH 7.9, 25% glycerol, 400 mM KCl, 1.5 mM MgCl 2, 0.2 mM EDTA, 0.5). mM dithiothreitol, 1 mM NaF, 1 mM sodium vanadate). The double-stranded NF-κB oligonucleotide (5-AGTTGAGGGGACTTTCCCAGGC-3, underlined with 10 μg of the nucleoprotein labeled [-32P] dATP) is underlined by the κB of the NF-κB / cRel homodimeric or heterodimeric complex. consensus sequence). The binding reaction was performed for 30 minutes at 37 ° C. with 30 μl of reaction buffer (10 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1 mM EDTA, 4% glycerol, 1 ug dI-dC, 1 mM dithiothreitol). The specificity of the binding was confirmed by comparison with an 80-fold unlabeled oligonucleotide. Separation of the DNA-protein conjugate with the unbound DNA probe was performed at a voltage of 100 V in a 5% native polyacrylamide gel using 0.5 × TBE buffer. The gel was vacuum dried at 80 ° C. for 1 hour and then exposed to X-ray film at −70 ° C. for 24 hours.

As a result, LPS-activated RAW 264.7 macrophages treated with 10, 30 and 60 µg / ml of KPR-2 showed inhibition of NF-κB DNA binding activity in the extract of the nucleus. The extent of this reduction was shown to be similar to that of iNOS and COX-2 proteins and mRNA.

The preparation examples of the pharmaceutical composition comprising the thawed bark, browned and lacquer extract of the present invention will be described, but the present invention is not intended to limit the present invention but is only intended to be described in detail.

Formulation example  One. Powder  Produce

Dry powder of KPR-2 ... ........... 300 mg

Lactose ... 100 mg

Talc .................. 10 mg

The above ingredients are mixed and filled in an airtight cloth to prepare a powder.

Formulation example  2. Preparation of Tablets

Dry powder of KPR-2 ... 50 mg

Corn Starch ............. 100 mg

Lactose ... 100 mg

Magnesium Stearate ... ...... 2 mg

After mixing the above components, tablets are prepared by tableting according to a conventional method for preparing tablets.

Formulation example  3. Manufacture of capsule

Dry powder of KPR-2 ... 50 mg

Corn Starch ............. 100 mg

Lactose ... 100 mg

Magnesium Stearate ... ...... 2 mg

According to a conventional capsule preparation method, the above ingredients are mixed and filled into gelatin capsules to prepare capsules.

Formulation example  4. Preparation of Injectables

Dry powder of KPR-2 ... 50 mg

Sterile Distilled Water for Injection ... ......... suitable

pH Adjuster ... ...... suitable

According to the conventional method for preparing an injection, the amount of the above ingredient is prepared per ampoule (2 ml).

Formulation example  5. Liquid  Produce

Dry powder of KPR-2 ... ........... 100 mg

Isomerized sugar ... 10 g

Mannitol ... 5 g

Purified water................................................. .................

After dissolving each component in purified water according to the usual method of preparing a liquid solution, adding lemon flavor appropriately, mixing the above components, adding purified water, adjusting the whole to 100 ml by adding purified water, and then filling into a brown bottle. The solution is prepared by sterilization.

Formulation example  6. Manufacture of healthy food

Dry powder of KPR-2 ... .......... 1000 mg

Vitamin Blend ... ........... suitable

Vitamin A Acetate 70 μg

Vitamin E ...................................... ............... 1.0 mg

Vitamin B1 ... 0.13 mg

Vitamin B2 ..................... ............. 0.15 mg

Vitamin B6 ... 0.5 mg

Vitamin B12 ... ............. 0.2 μg

Vitamin C ... ............... 10 mg

Biotin ... 10 μg

Nicotinic Acid Amide ... ......... 1.7 mg

Folic Acid ... ... 50 μg

Calcium Pantothenate ... 0.5 mg

Mineral Mixture ... ........... suitable

Ferrous Sulfate ............. ............... 1.75 mg

Zinc Oxide ... ............. 0.82 mg

Magnesium Carbonate ... ... 25.3 mg

Potassium monophosphate ........................ 15 mg

Dibasic calcium phosphate ..... 55 mg

Potassium Citrate ... .............. 90 mg

Calcium Carbonate ... ............... 100 mg

Magnesium Chloride ... .......... 24.8 mg

Although the composition ratio of the above-mentioned vitamin and mineral mixtures is mixed with a component suitable for a health food in a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional health food manufacturing method. The granules may be prepared and used for preparing a health food composition according to a conventional method.

Formulation example  7. Manufacture of health drinks

Dry powder of KPR-2 ... ......... 1000 mg

Citric Acid ... .............. 1000 mg

oligosaccharide................................................. .............. 100 g

Plum concentrate ..................... ............... 2 g

Taurine ... ......... 1 g

Purified water is added to the whole ........... .... 900 ml

After mixing the above components according to a conventional healthy beverage manufacturing method, and then stirred and heated at 85 ℃ for about 1 hour, the resulting solution is filtered and obtained in a sterilized 2 L container, sealed and sterilized and then refrigerated and stored in the present invention For the preparation of healthy beverage compositions.

Although the composition ratio is mixed with a component suitable for a favorite beverage in a preferred embodiment, the composition ratio may be arbitrarily modified according to regional and ethnic preferences such as demand hierarchy, demand country, and usage.

As described above, the thawed bark, browned and lacquer extract of the present invention or a mixed extract thereof has an anti-inflammatory effect, and thus can be used as a pharmaceutical composition and health functional food for the prevention and treatment of inflammation-related diseases.

Claims (1)

A pharmaceutical composition effective in the prevention and treatment of inflammation caused by TNF-α and PGE2 over-formation containing thaw skin, browning and water or ethyl acetate soluble extract of lacquer as an active ingredient, and containing a pharmaceutically acceptable carrier or excipient.

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