KR20040075135A - Composition comprising the extract of Saururus chinensis having anti-inflammatory activity - Google Patents

Abstract

PURPOSE: A composition including a Saururus chinensis extract as an effective ingredient and a pharmaceutically acceptable carrier or excipient is provided. It significantly inhibits NO and PGE2 which intermediate an inflammatory mechanism and markedly suppress the expression of induction type NOS(iNOS), COX-2 protein and mRNA. CONSTITUTION: The pharmaceutical composition having an effect of preventing and treating an inflammation-related disease contains 0.1 to 80% by weight of a Saururus chinensis extract and a pharmaceutically acceptable carrier or excipient. A health supplementary food exhibiting preventing effect on the inflammation-related disease contains the Saururus chinensis extract and a food acceptable food supplementary additive and is in the shape of powder, granules, tablets, capsules and beverages.

Description

Composition comprising the extract of Saururus chinensis having anti-inflammatory activity

The present invention relates to a three hundred sec extract having anti-inflammatory activity and a composition containing the same, to a pharmaceutical composition and health functional food for the prevention and treatment of 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, inflammatory reactions restore the function of life by removing external infectious agents and regenerating damaged tissues.However, if the inflammatory response is excessive or persistent due to the absence of antigens or internal substances, the major pathology of the disease (sensitizing disease) , Chronic inflammation) and also become a disorder in the treatment process such as blood transfusion, drug administration, and organ transplantation.

In vivo, inflammation causes various biochemical phenomena. Especially, nitric oxide synthase (NOS) and prostaglandin, enzymes that generate nitric oxide (NO), are involved. Enzymes related to biosynthesis are known to play an important role in mediating the inflammatory response, thus synthesizing prostaglandins from NOS, an enzyme that produces NO from L-Arginine, or Arachidonic acid. COX (cyclooxygenase), an enzyme that is involved in the detoxification, is a major target in blocking inflammation.

Recent studies have shown that there are several types of NOS, such as brain NOS (brain NOS) in the brain, nNOS (neuronal NOS) in the nervous system, and endothelial NOS (eNOS) in the vascular system. While small amounts of NO produced by these cells play an important role in maintaining normal homeostasis, such as inducing neurotransmission and vasodilation, iNOS (induced by various cytokines or external stimulants) NO, which is excessively produced by induced NOS, is known to cause cytotoxicity and various inflammatory reactions, and chronic inflammation has been associated with an increase in iNOS activity (Miller MJ et al ., Mediators of inflammation , 4 , pp387). -396, 1995: Appleton L. et al ., Adv. Pharmacol. , 35 , pp 27-28, 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, relate to the pathogenesis of chronic diseases including various sclerosis, parkinson's disease, Alzheimer's disease and colon cancer.

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, but it 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), pp 2423-2432, 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), pp 311-320, 1998: Landry, DB et al ., Am. J. Pathol. , 151 (4), pp 1085-1095, 1997: Gerritsen, ME et al ., Am. J Pathol. , 147 (2), pp 278-292, 1995).

Therefore, the preferred anti-inflammatory agent should be a substance having a selective inhibitory effect on iNOS or COX-2, and the most ideal anti-inflammatory agent is selective to iNOS and COX-2 and can be said to inhibit both.

NOS inhibitors known to date include L-NMMA (NG-methyl-L-arginine), L-NNA (NG-nitro-L-arginine), L-NAME (NG-nitro-L-arginine methyl ester), aminoguanidine (Aminoguanidine), N-thioureido-L-norvaline, and S-methyl-L-thiocitrulline, but most of them are associated with L-arginine, a substrate of NOS. It acts as an inhibitor of NOS and therefore has the disadvantage of not being selective for iNOS.

Recently, NS-398, CGP-29238, SC-58125, L-745337, meloxicam, etc. have been reported as inhibitors for COX-2 produced by synthetic methods, but are not yet widely used. Their side effects are not well proven.

Three hundred scent (Sarurus chinensis ) used in the present invention is a perennial herb belonging to the three hundreds (Saururaceae) in the folk horticulture (whole grass) or root, leaf is poisonous (風 毒), diuretic (利尿), species (水腫) , Gonorrhea, hepatitis, pneumonia, venom, hypertension, etc., and the components of the outpost are methyl- n -nonyl-ketone n -nonyl-ketone, stems contain hydrolysable tannins, leaves contain quercetin, quercitrin, isoquarcitrin, abiculin, ahyperin, It contains rutin and hydrolyzable tannins (Bo Bo-seop and Shin Mingyo, Ph.D., Yeonglimsa, pp812-814, 1990).

On the other hand, in natural plants, few studies have been conducted except that some plants and other extracts have been reported to have inhibitory activity against iNOS or COX-1, and moreover, selective inhibition of iNOS and COX-2 simultaneously. Therefore, there is no report on the substance applicable as an anti-inflammatory agent. In addition, the function as an antioxidant of three hundred seconds used in the present invention is already known (Korean Patent Publication No. 10-2000-0029632), but no information on anti-inflammatory has been reported.

The inventors of the present invention studied the effect on the anti-inflammatory activity of the extract of three hundred seconds through the molecular biological experiments as well as microscopic observation through cell line cultures, iNOS, COX-2 in the LPS-induced macrophages of three hundred seconds extract The present invention was completed by inhibiting enzyme and mRNA expression and inhibiting DNA binding activity of inflammatory transcription factor NF-κB and its subunit IκB degradation.

The present invention relates to a three hundred sec extract having anti-inflammatory activity and a composition containing the same, to provide a pharmaceutical composition and health functional food for the treatment and prevention of various inflammation-related diseases.

1 is a diagram showing the effect on nitric oxide (NO) production by three hundred seconds extract in RAW 264.7 macrophages induced with lipopolysaccharide (LPS),

Figure 2 is a diagram showing the effect on the production of prostaglandin E ₂ (PGE ₂ ) by three hundred seconds extract in LPS-induced RAW 264.7 macrophages,

Figure 3a is a Western blot analysis showing the expression regulation of inducible nitric oxide synthase (iNOS) enzyme by three hundred seconds extract in LPS-induced RAW 264.7 macrophages,

Figure 3b is a result of RT-PCR showing the effect of iNOS mRNA expression by three hundred seconds extract in LPS-induced RAW 264.7 macrophages,

Figure 4a is a Western blot analysis showing the expression regulation of COX-2 (cyclooxygenase-2) enzyme by trichophytium extract in LPS-induced RAW 264.7 macrophages,

Figure 4b is a result of RT-PCR showing the effect of COX-2 mRNA expression by three hundred sec extract in LPS-induced RAW 264.7 macrophages,

5 is a diagram showing the inhibition of COX-2 enzymatic activity of NS-398 extract and control drug in LPS-derived RAW 264.7 macrophages,

Figure 6 is a diagram showing the inhibition of NF-κB DNA binding, which is an inflammatory transcription factor according to the concentration of three hundred seconds extract,

Figure 7a is a Western blot analysis showing the degradation of IκB according to the concentration of three hundred seconds extract,

Figure 7b is a Western blot analysis showing the nuclear translocation of p65 protein according to the concentration of three hundred seconds extract.

In accordance with the above object, the present invention provides a pharmaceutical composition effective to the prevention and treatment of inflammation-related diseases containing three hundred s. Extract as an active ingredient, and containing a pharmaceutically acceptable carrier or excipient.

The inflammation related diseases include pain such as acute pain, chronic pain, neuropathic pain, postoperative pain, migraine and joint pain; Fever in neuropathy, nerve damage, stroke, irritable bowel syndrome, asthma and chronic obstructive pulmonary disease; Gastric-duodenal ulcer; And inflammatory bowel disease.

The composition for the prevention and treatment of inflammation-related diseases of the present invention, the total weight of the composition comprises 0.1 to 80% by weight, preferably 0.5 to 50% by weight of the extract.

Method of separating the three hundred seconds extract of the present invention is as follows.

The triticale extract of the present invention is about 1 to 10 times, preferably about 2 to 5 times the volume of water, lower alcohol or about 1: 0.1 to 1:10, preferably about 3 to 300 times the weight of dried triticale famine. Is a mixed solvent having a mixing ratio of 1: 0.2 to 1: 3, ultrasonic extraction for about 0.5 hours to 2 days, preferably 1 hour to 1 day at an extraction temperature of 20 to 100 ℃, preferably 60 to 80 ℃, An extraction method such as reflux extraction may be repeated once to 10 times, preferably 3 to 7 times, to obtain an extract that is soluble in water, lower alcohols, or a mixed solvent thereof.

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

Compositions comprising extracts according to the invention are formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories and sterile injectable solutions, respectively, according to conventional methods. Can be used. Specifically, when formulated, it may be prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, and the like. 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 can be mixed. In addition to simple excipients, lubricants such as magnesium stearate, talc can also be used. Liquid preparations for oral use may include various excipients such as wetting agents, sweeteners, fragrances, preservatives, etc., in addition to water and liquid paraffin, which are simple diluents commonly used in suspensions, solutions, emulsions, and syrups. have. 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.

The extract of the present invention may vary depending on the age, sex, and weight of the patient, but may generally be administered in an amount of 0.01 to 500 mg / kg, preferably 0.1 to 100 mg / kg, divided once or several times daily. . In addition, the dosage of the extract can be increased or decreased depending on the route of administration, the degree of disease, sex, weight, age and the like. Therefore, the above dosage does not limit the scope of the present invention in any aspect.

Since the extract of the present invention has little toxicity and side effects, it can be used safely even when taken for a long time for the purpose of prevention.

The present invention provides a health functional food comprising a three hundred sec extract and a food acceptable acceptable food supplement additive exhibiting a prophylactic effect of inflammation-related diseases.

The composition containing the extract of the present invention can be used in various ways, such as drugs, foods and drinks for the prevention of inflammation-related diseases. Foods to which the extract of the present invention can be added include various foods, for example, beverages, gums, teas, vitamin complexes, and health functional foods, and can be used in the form of powders, granules, tablets, capsules or beverages. .

In this case, the amount of the extract in the food or beverage, in general, may be added to 0.01 to 15% by weight of the total food weight in the case of the health food composition of the present invention, 0.02 to 10g based on 100ml, preferably in the health beverage composition It may be added at a ratio of 0.3 to 1g.

The health beverage composition of the present invention has no particular limitation on the liquid component except for containing the extract as an essential ingredient in the indicated ratio, 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; Polysaccharides such as dextrin, cyclodextrin; Conventional sugars such as and the like and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those described above, natural flavoring agents (tauumatin, stevia extract (e.g., Rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. have. The proportion of such natural carbohydrates is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the composition of the present invention.

In addition to the above, the composition 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 salts thereof. , Organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. In addition, the compositions of the present invention may contain fruit flesh for the production of natural fruit juices and fruit juice drinks and vegetable drinks. These components can be used independently or in combination. The proportion of such additives is not so critical but is generally selected from the range of 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

The present invention is described in more detail based on the following examples and experimental examples, but the present invention is not limited thereto.

Reference Example 1. Laboratory Instruments and Reagents

RPMI 1640 medium, fetal bovine serum (FBS), penicillin and streptomycin were purchased from Life Technologies Inc., USA, and COX-2, iNOS, NF-κB and IκBα monoclonal antibodies and peroxidases Conjugated secondary antibodies were purchased from Santa Cruz Biotechnology, Inc., USA. EIA kits for measuring prostaglandin E ₂ were purchased from R & D Systems (USA) and NS398, COX-2 enzyme inhibitors were purchased from Calbiochem (USA). RNA extraction kits include Intron biotechnology (Intron biotechnology, Korea); iNOS, COX-2, GAPDH oligonucleotide primers were purchased from Bioneer (Bioneer, Korea). 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl-tetrazolium bromide (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl-tetrazolium bromide, MTT), 3- (2-hydroxy-2-nitroso-1-propylhydrazino) -1-propanamine (3- (2-hydroxy-2-nitroso-1-propylhydrazino) -1-propanamine, PAPA NONOate, sulfanilamide, aprotinin, leupeptin, phenylmethylsulfonyl fluoride (PMSF), dithiothreitol, caffeic acid, N ^G- M-L-arginine (N ^G -methyl-L-arginine, NMMA), LN ^6- (1-iminoethyl) lysine (LN ^6- (1-iminoethyl) lysine, L-NIL), E.coli lipopolysaccharide (lipopolysaccharide, LPS) and all other chemicals were purchased from Sigma (USA).

Example 1. Preparation of three hundred vine extract

The famine of three hundred seconds was collected from a medicinal plant farm (Sambu Farm, Geochang-si) in May 1998 and verified by Dr. Ahn Byung-tae of Pharmacy at Chungbuk National University. .

70 g of dried three hundred scents were selected and chopped. Then, 2 liters of methanol solution was added, and the mixture was refluxed three times at 70 ° C. and filtered through a filter paper (Whatman, USA). Concentration and lyophilization gave 7.1 g of extract, which was used as a sample.

Experimental Example 1. Nitrite Assay

In order to confirm the nitric oxide inhibitory activity of the extract of three hundred seconds of the present invention, the following experiment was performed.

Nitrite accumulation, a marker of NO synthesis, was measured in the medium by a griess reaction. RAW 264.7 macrophages (Korea Cell Line Bank) were cultured in RPMI medium containing 10% FBS, penicillin (100 units / ml) and streptomycin sulfate (100 µg / ml) at 37 ° C., 5% carbon dioxide, and 300 seconds. Extracts 1, 5, 10 μg / ml and lipopolysaccharide (LPS) were treated and incubated for 24 hours. 100 μl of cell medium was added to Gries reagent (5% (v / v)) (the same amount of 1% (w / v) sulfanylamide dissolved in phosphoric acid and 0.1% (w / v) naphthylethylenediamine-hydrochloric acid) 100 After mixing in μl, the cells were incubated for 10 minutes at room temperature, and then absorbance was measured at 550 nm using a microplate reader (Power Wave 340, Bio-Tek, USA). Fresh culture medium was used as a non-treatment group in all experiments, LN ⁶ - (1- Iminoethyl) lysine ^{(LN 6 - (1-iminoethyl} ) lysine, L-NIL) 10μM was used as the positive inhibitor. The amount of nitric oxide in the sample was calculated from the sodium nitrite standard curve prepared in the medium, and the data were expressed as mean ± standard deviation by three independent measurements, all experiments being at least three times, each three The analysis was conducted by one or more independent observations, and statistical analysis was performed by Student's test.

As a result, the LPS-induced NO ^2- producing was about 10-fold, and it was confirmed that NO ^2- producing was inhibited by dose-dependent administration of the 300 sec extract (see FIG. 1).

Experimental Example 2. Prostaglandin E ₂ (PGE ₂ ) exam

In order to confirm the PGE ₂ inhibitory activity of the extract of the three hundred seconds of the present invention, the following experiment was performed.

The same cell culture as in Experimental Example 1 was carried out, and the cells were pre-cultured with 300 seconds extract for 1 hour, and then activated with 1 μg / ml of LPS. PGE ₂ measurements in macrophage medium were quantified with an EIA kit according to the manufacturer's instructions.

As a result, it was found that the extract of three hundred seconds significantly inhibited PGE ₂ production, and in particular, it was confirmed that PGE ₂ production was significantly inhibited (see FIG. 2).

Experimental Example 3. iNOS Inhibitory Activity Test

In order to confirm the iNOS inhibitory activity of the three hundred seconds extract of the present invention, the following experiment was performed.

3-1. Western blot test

Cellular proteins were extracted from RAW 264.7 cells treated with control and three hundred s crude extracts. Cells were collected by centrifugation, washed once with phosphate buffered saline, and washed cells (50 mM HEPES pH 7.0, 250 mM sodium chloride, 5 mM EDTA, containing 5 μg / μl of leupetin and aprotin, respectively). 0.1% Nonniche P-40, 1 mM phenylmethylsulfonyl fluoride, 0.5 mM dithiothreitol, 5 mM sodium fluoride, 0.5 mM sodium orthovanadate) and incubated at 4 ° C. for 15 minutes. Cell debris was removed by microcentrifuge and rapidly frozen the supernatant. Protein concentrations were determined using protein test reagents according to the manufacturer's (Bio-Rad) instructions. 50 μg of cellular proteins obtained from treated and untreated cell extracts were electrophoresed with nitrocellulose membranes, followed by separation of 8-12% SDS-polyacrylamide gels. Immunoblots were incubated overnight at 4 ° C. with blotting solution (5% skim milk) and incubated for 4 hours with 1: 500 dilution of single antibody anti-iNOS. Blots were washed twice with Tween 20 / Tris buffer saline (TTBS) and incubated for 1 hour at room temperature with 1: 1000 dilution of horseradish peroxidase-conjugated goat anti mouse IgG secondary antibody. Blots were again washed three times with TTBS and measured with enhanced chemiluminescence (Chemiluminescence, Amersham Life Science, USA).

As a result, the expression of iNOS was significantly increased by LPS, and it was confirmed that iNOS protein induction was highly inhibited depending on the concentration of trichophytium extract 1, 5, and 10 µg / ml (see FIG. 3A).

3-2. RNA Preparation and Polymerase Chain Reaction (RT-PCR)

Total cell RNA was isolated using Easy Blue kits (Intron Biotechnology, South Korea) according to the manufacturer's instructions. 1 μg of RNA from each sample was reverse transcribed using MuLV reverse transcriptase, 1 mM dNTP, and oligo (dT _12-18 ) 0.5 μg / μL, followed by a thermal cycler (Perkin Elmer Cetus). PCR analysis was performed using cDNA purification to search for iNOS and GAPDH gene expression. The reaction was performed in 25 μl containing 2 units of Taq DNA polymerase, 0.2 mM dNTP, × 10 reaction buffer, 100 pM of 5 ′ and 3 ′ primers. After initial denaturation at 95 ° C. for 2 minutes, the cycle number was used within the exponential range of iNOS response to iNOS (1 minute at 95 ° C., 1 minute at 60 ° C., 1.5 minutes at 72 ° C.). PCR was electrophoresed on 2.5% agarose gel and stained with ethidium bromide and detected by UV.

As a result, it was confirmed that iNOS mRNA showed a significant dose-dependent decrease in treatment with 1, 5, and 10 µg / ml of three hundred sec extract (see FIG. 3B).

Experimental Example 4. COX-2 Inhibitory Activity Test

In order to confirm the COX-2 inhibitory activity of the three hundred seconds extract of the present invention, the following experiment was performed.

4-1. Western blot test

The experiment was performed in the same manner as in Experiment 3-1. Immunoblot was incubated overnight at 4 ° C. with a blotting solution (5% skim milk), for 4 hours with a 1: 500 dilution of COX-2 antibody. Cultured and enhanced chemifluorescence measurement.

As a result, the degree of COX-2 expression was significantly increased by LPS, and it was confirmed that COX-2 protein expression induced by LPS was inhibited by 68.5% when treated with 10 µg / ml of 300 seconds extract. In addition, it was confirmed that the 300 seconds extract 1, 5, 10 ㎍ / ㎖ treatment inhibits COX-2 protein induction in a concentration-dependent (see Figure 4a).

4-2. RNA Preparation and Polymerase Chain Reaction (RT-PCR)

In the same manner as in Experiment 3-2, PCR analysis was performed by cDNA purification to detect COX-2 and GAPDH gene expression. After the initial denaturation, the cycle number was used within the exponential range for COX-2 (1 min at 94 ° C., 1 min at 60 ° C., 1 min at 72 ° C.) and electrophoresed on 2.5% agarose gel, Staining with ethidium bromide was detected by UV.

As a result, it was confirmed that COX-2 mRNA showed a significant dose-dependent decrease in treatment with 1, 5, and 10 µg / ml of three hundred sec extract (see FIG. 4B).

Experimental Example 5. COX-2 Inhibitory Activity Test Method

In order to confirm the COX-2 inhibitory activity of the three hundred seconds extract, the following experiment was performed.

RAW 264.7 cells were placed in 2 × 10 ⁵ cells / well in 24 wells, each incubated with glycolipids for 6 hours, and then washed twice with PBS. To measure COX-2 activity, after washing with fresh medium twice the cells were equilibrated for 30 minutes with or without three hundred seconds extract sample. Cells were incubated in medium with 100 μM arachidonic acid that had not been treated with LPS for 15 minutes, and the supernatant was removed and PGE ₂ was measured using the enzyme immunoassay kit. NMMA and NS-398 were used as controls.

Experimental results showed that 1, 5, and 10 µg / ml extracts did not show significant inhibition on COX-2 enzyme activity in RAW 264.7 macrophages, whereas NS-398, a 50 μM specific inhibitor of COX-2 activity, was COX-2. Inhibition of enzyme activity inhibited 63.8% of PGE ₂ production (see FIG. 5).

Experimental Example 6. Removal effect of PAPANONOate-released NO radicals

In order to confirm the NO radical removal effect of the three hundred seconds extract of the present invention, the following experiment was performed.

PAPA NONOate, a water-soluble NO / nucleophilic complex, can automatically separate pH-dependent free amines and NO radicals at the first rate of reaction (Morley D. et al ., J. Cardiovasc. Pharmacol. , 22 , pp235-246, 1998), PAPA NONOate One molecule generates two NO molecules.

20 μM of PAPA NONOate was dissolved in RPMI 1640 medium (pH 7.6) containing serum prepared for cell culture, and 1, 5, and 10 μg / ml of three hundred seconds extracts were incubated at 37 ° C. for 3 hours. After incubation, the concentration of NO was measured using the Gries method, and caffeic acid was used as a positive control.

Experimental results showed that 100 μM caffeic acid used as a positive control significantly inhibited NO radicals from 36.53 ± 0.24 μM to 13.59 ± 0.47 μM, whereas 1, 5, and 10 μg / ml extracts of 300 sec showed NO radicals. Did not remove Eventually, the extract of 300 sec did not directly interact with NO, and was found to inhibit NO and PGE ₂ production by reducing iNOS and COX-2 protein and mRNA expression.

Experimental Example 7. Analysis of Electrophoretic Mobility Shift Assay (EMSA)

In order to identify the iNOS and COX-2 transcription inhibition mechanism mediated by the extract of Triticalis, experiments were performed using these well-known transcription factors as follows.

In unstimulated cells, NF-κB was isolated from the cytosol by LPS stimulation of the inhibitor IκB, and LPS stimulation of IκB was phosphorylated by the inhibitor, IκB kinase, binds and rapidly forms 26s proteases. It is degraded through proteosomes and thus releases NF-κB (Baeuerle PA, Cell , 95 , pp729-731, 1998).

RAW 264.7 macrophages were placed in 100 mm dishes (5 ± 10 ⁶ cells), treated with 300 sec extract 1, 5, 10 μg / ml, stimulated with LPS for 1 hour, washed once, and then cold PBS solution 1 Collected in ml, pellets were prepared by centrifuge. Cytosolic and nuclear fractions were extracted as above (Liu GY et al ., Mol. Carcinog ., 22 , pp235-246, 1998). The binding reaction was 30,000 cpm ³² P for 10 mM tris-hydrochloric acid, pH 7.5, 50 mM sodium chloride, 1 mM EDTA, 10% glycerol, 1 μg poly (dI-dC), 1 mM dithiothreithol and NF-κB. -15 [deg.] C. was carried out in 20 [mu] l of reaction buffer containing labeled oligonucleotide probe for 15 minutes. DNA-protein complexes were isolated from unbound DNA probes on 80 volt native 5% polyacrylamide gels in 0.5 × TBE buffer, and the gels were vacuum dried at 80 ° C. for 1 hour and at 70 ° C. for 24 hours. X-ray film was exposed.

Nuclear extracts obtained from LPS-activated RAW 264.7 macrophages treated with 1, 5 and 10 µg / ml concentrations of 300 sec extracts showed a significant decrease (70%) in NF-κB DNA binding activity. (See FIG. 6).

Treatment of 10 μg / ml of Trichophyll extract showed that it did not affect the degradation of IκBα constituting the IκB complex in the cytosol until 180 minutes (see FIG. 7A), after the trichophytic extract was released from IκB As a result of confirming that it interferes with the translocation of NF-κB and p65 subunits from the cytosol to the nucleus, it was confirmed that LPS-induced p65 protein in the nucleus was significantly reduced by the extract of three hundred seconds (FIG. 7B). Reference).

Experimental Example 8. Cytotoxicity Test by MTT

In order to confirm the cytotoxic effect of the three hundred seconds extract obtained in Example 1 of the present invention, the following experiment was performed.

The mouse macrophage RAW 264.7 (Korea Cell Line Bank) was used in a humid environment of 37 ° C and 5% carbon dioxide in RPMI medium containing 10% FBS, penicillin (100 units / ml) and streptomycin sulfate (100 µg / ml). Incubated. RAW 264.7 cells were collected with a scraper and placed in 1 × 10 ⁵ / well in a 96 well plate containing 100 μl of RPMI 1640 medium containing 10% FBS. The tricot sec crude extract was dissolved in DMSO solvent and the concentration of DMSO in all tests did not exceed 0.1%. After overnight, samples were added and the plates incubated for 24 hours. After washing the cells once, 50 μl of FBS-free medium containing 5 mg / ml MTT was added, and after 4 hours of incubation at 37 ° C., the medium was removed and the formazan blue formed in the cells was removed. After dissolving in 100 μl of DMSO, absorbance was measured at 540 nm.

As a result, even in the presence of three hundred seconds alone and lipopolysaccharide could not observe any effect on the survival of RAW 264.7 cells for 24 hours, it was confirmed that the three hundred seconds extract is a safe drug.

The three hundred s. Extract of the present invention and the composition containing the same may be administered in the following formulations, and the following formulation examples are merely illustrative of the present invention, thereby not limiting the content of the present invention.

Formulation Example 1 Preparation of Injection

100 mg of extract of Example 1

Sodium Metabisulfite 3.0 mg

Methylparaben 0.8 mg

Propylparaben 0.1 mg

Appropriate sterile distilled water for injection

The above ingredients are mixed and prepared in a conventional manner to have a final volume of 2 ml, and then filled into 2 ml ampoules and sterilized to prepare an injection.

Formulation Example 2 Preparation of Tablet

200 mg of extract of Example 1

Lactose 100 mg

Starch 100 mg

Magnesium stearate proper amount

A tablet is prepared by mixing and tableting the above components according to a conventional tablet manufacturing method.

Formulation Example 3 Preparation of Capsule

100 mg of extract of Example 1

Lactose 50 mg

Starch 50 mg

Talc 2 mg

Magnesium stearate appropriate amount

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 Liquid

1000 mg of extract of Example 1

20 g of sugar

20 g of isomerized sugar

Lemon flavor

Purified water was added to make a total of 1000 ml. According to the conventional method for preparing a liquid, the above components are mixed, and then filled into a brown bottle and sterilized to prepare a liquid.

Formulation Example 5 Preparation of Healthy Food

1000 mg of extract of Example 1

Vitamin mixture proper amount

70 μg of Vitamin A Acetate

Vitamin E 1.0 mg

Vitamin B ₁ 0.13 mg

Vitamin B ₂ 0.15 mg

Vitamin B ₆ 0.5 mg

0.2 μg of vitamin B ₁₂

Vitamin C 10 mg

10 μg biotin

Nicotinic Acid 1.7 mg

Folate 50 ㎍

Calcium Pantothenate 0.5mg

Mineral mixture

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 6 Preparation of Healthy Drink

1000 mg of extract of Example 1

Citric acid 1000 mg

100 g oligosaccharides

Plum concentrate 2 g

1 g of taurine

Add 900 ml of purified water

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

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

The triticale extract of the present invention and the composition containing the same significantly inhibit NO and PGE ₂ mediating inflammatory mechanisms, and pain by confirming that significantly inhibit the expression of induced NOS (iNOS), COX-2 protein and mRNA And it can be usefully used for the treatment and prevention of various inflammatory diseases.

Claims (5)

A pharmaceutical composition containing the extract of Triticales as an active ingredient and effective for the prevention and treatment of inflammation-related diseases containing a pharmaceutically acceptable carrier or excipient. The method of claim 1, wherein the inflammation-related disease is acute pain, chronic pain, neuropathic pain, postoperative pain, pain such as migraine and joint pain; Fever in neuropathy, nerve damage, stroke, irritable bowel syndrome, asthma and chronic obstructive pulmonary disease; Gastric-duodenal ulcer; Or inflammatory bowel disease. The pharmaceutical composition of claim 1, wherein the extract comprises 0.1 to 80% by weight. A health functional food comprising a extract of 300 sec and a food acceptable acceptable food supplement, which show a prophylactic effect of an inflammation-related disease. The dietary supplement of claim 4 in the form of a powder, granule, tablet, capsule, beverage.