KR101264293B1 - Composition for Treating or Preventing Inflammatory Diseases or Oxidatvie Damage Associated Diseases Comprising the Extraction of Bambusae Caulis in Liquamen - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for the treatment or prophylaxis of inflammatory diseases or diseases caused by oxidative damage, including the extract as an active ingredient. The bamboo extract of the present invention and the active compound 1, 2-dihydroxybenzene (1, 2-dihydroxybenzene) or 1, 4-dihydroxybenzene (1, 4-dihydroxybenzene) separated therefrom are nitrogen oxides (Nitric Oxide, By having NO) inhibitory activity and free radical scavenging ability, it can be developed as a medicine for the treatment or prevention of inflammatory diseases or diseases caused by oxidative damage by free radicals.

Pulmonary extract, anti-inflammatory activity, antioxidant activity, NO (Nitric Oxide), inflammatory disease, disease caused by oxidative damage

Description

Composition for Treating or Preventing Inflammatory Diseases or Oxidatvie Damage Associated Diseases Comprising the Extraction of Bambusae Caulis in Liquamen}

The present invention relates to a pharmaceutical composition for the treatment or prophylaxis of inflammatory diseases or diseases caused by oxidative damage, including the extract as an active ingredient. The present invention also relates to a functional food composition for amelioration or alleviation of diseases caused by inflammatory diseases or oxidative damage, which contains the extract of bamboo as an active ingredient.

Bamboo (Bambusae Caulis in Liquamen, BCL) is a distillate obtained by heating bamboo at high temperature. It is produced using Gramineae's native cotton (Phyllostachys nigra var. Henosis), which is native to Korea. Bamboo has been known as bamboo juice and bamboo water for many years.In civilian cough, asthma, hypertension, ischemic heart disease, stroke, diabetes, athlete's foot, burn treatment, digestive disorders, etc. come. ^1-3)

According to a recent study on the composition of the killing force, the extract analyzed by gas chromatography contained 80.76% acetic acid, 4.60% ethanol, 2.49% p-cresol, dimethyl Phenol (dimethyl phenol) 2.35%, p-isopropyl phenol 2.00%, propanol, hydroxybuthane 1.33%, nitrophenol 0.81%, propionic acid, It has been reported that 0.76% o-cresol, 0.46% methanol and more than 300 alcohols, aldehydes and phenol derivatives. ^4-6)

A study on the tabasheer bioactive 1982 Hong et tabasheer extract analgesia, intestinal smooth muscle relaxation, blood pressure lowering effect, was reported to be in Jinhae acts, ^7), such as Kim 1998 tabasheer extract induced ischemic heart disease In one mouse model, heart rate, left ventricular pressure, and coronary blood flow rate were restored, and lactate dehydrogenase (LDH) and creatine phosphokinase (CPK) were reduced, resulting in myocardial protection. ^{8) In} 1999, Chung et al reported that T-lymphocytes, T-lymphocytes in the spleen, and macrophage in the peritoneum were reduced in rats fed with water of death. We have reported immunomodulatory activity with increased macrophage activity. ^In addition, in 2001, Jang et al. Reported the results of the study that kill extract reduced blood glucose levels in the mouse diabetic model induced by streptozotocin (Streptozotocin), and ⁶⁾ Chung et al. The hypoglycemic effect is explained by the study that the insulin-immunoreactivity of islet pancreatic beta cells is restored. ^{5) In} 2002, Chung et al. Demonstrated that liver and kidney function declined in rats fed daily dietary force (1: 3 = BCL: water concentration) for 24 weeks. The toxicity of death was reported.10 ^{) In} 2002, Park et al. Administered oral and saliva X mice to alcohol-administered rats. It recovers effects reported among which the reduction ^{dismutase) 11)} 2003 years ago tabasheer the 3T3-L1 bulb fat cells (preadipocytes) and to assist the growth of the cells synthesize fat cells (adipocytes) type IV collagen (collagen) in The study reported that it makes D more denser. ^{12) In} 2004, Choi et al. Demonstrated that pulmonary extracts inhibit antioxidant activity and HMG-CoA (3-hydroxy-3-methylglutaryl coenzyme A) activity. It has been shown to promote lipid metabolism by reducing plasma total cholesterol, low density lipoprotein-cholesterol, triglyceride, and increasing high density lipoprotein-cholesterol and phospholipid. ^{13) In} 2004, Kim et al. Reported that mixed extracts of root and death increased AST (Aspartate aminostransferase) and PAS (Mn-SOD periodic acid schiff) -positive responses in alcohol-ingested rats and repaired hepatocyte damage. was reported the possibility of a hangover drink, ¹⁴⁾ in 2005 is positive-positive bacteria, such as the X tabasheer of water and 50% ethanol x gram (gram positive bacteria; Bacill We report the antimicrobial activity of death that inhibits the growth of us subtilis, Listeria monocytogenes) and gram negative bacteria (Sigella dysenteriae). ¹⁵⁾

Nitric oxide (NO) is synthesized by nitric oxide synthase (NOS) from L-arginine in various tissues and cells, and is known as vasodilation, neurotransmission, blood coagulation, and immune function. It is known to play a role in regulation. NOS can be divided into two groups, cNOS and iNOS. In other words, neuronal, endothelial NOS belongs to cNOS, which is Ca ²⁺ -calmodulcin-dependent and normally secretes NO continuously to regulate homeostasis. Inducible NOS (iNOS), on the other hand, is activated by cytokines such as IFN-γ, interleukin-1, and tumor necrosis factor-α, or by lipopolysaccharide (LPS) of bacteria. Will be created. This NO plays a major role in defending the host from microorganisms or tumor cells because it has a large effect on the cytotoxic activity of macrophages. However, over-produced NO exacerbates the inflammatory response, such as rheumatoid arthritis, and thus exhibits harmful effects. ^22,23) Stimulated iNOS is present in macrophage and hepatocytes, while NO production is markedly increased and cytotoxic while inflammation is involved with other pathogens. Furthermore, the large reaction of normal free radicals of NO with oxygen to produce nitrogen dioxide produces NO, a powerful pro-oxidant molcule that can cause loss of oxidative power. Therefore, inhibition of NO production in response to inflammatory stimuli can be used as a therapeutic method in inflammatory diseases. ^24,25)

Free radicals and other reactive oxygen species are produced by endogenous metabolites in the food or endogenous metabolism in the body. Radicals can oxidize biomolecules to cause oxidative damage leading to apoptosis or tissue damage. ³¹ Atherosclerosis, cancer, emphysema, cirrhosis and arthritis are known to be associated with oxidative damage. ^31,32) Thus, oxidative damage plays a very important pathological role in human disease, and the consumption of foods containing antioxidant supplements or antioxidants is of great importance because it reduces oxidative damage in the human body. ^{33 In} recent years, natural antioxidants in foods such as vitamins C, E, sesamol and carnosic acid have been provided to consumers.

DPPH (1,1-diphenyl-2-picrylhydrazyl) has the property of accepting hydrogen atoms easily because the nitrogen atom of hydrazyl is in an unstable state. Therefore, the degree of antioxidant capacity can be measured by using the property of reacting with an antioxidant and losing hydrogen color by accepting hydrogen atoms. ²⁶⁾

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors have tried to extract the active ingredient having an improved anti-inflammatory and antioxidant activity among various natural products, confirming that the Bambusae Caulis in Liquamen extract has anti-inflammatory and antioxidant activity, and active from the extract Compounds 1,2-dihydroxybenzene and 1,4-dihydroxybenzene separated by activity guided isolation are the nitric oxides. The present invention was completed by experimentally confirming that the formation inhibitory activity and the free radical scavenging ability have very excellent anti-inflammatory and antioxidant activity.

Accordingly, it is an object of the present invention to provide a composition for treating or preventing inflammatory diseases comprising Bambusae Caulis in Liquamen extract as an active ingredient.

Another object of the present invention is to provide a composition for treating or preventing diseases caused by oxidative damage caused by free radicals including Bambusae Caulis in Liquamen extract as an active ingredient.

The objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, the present invention provides a pharmaceutical composition comprising (a) a pharmaceutically effective amount of Bambusae Caulis in Liquamen extract; And (b) provides a pharmaceutical composition for the treatment or prevention of inflammatory diseases comprising a pharmaceutically acceptable carrier.

According to another aspect of the present invention, the present invention provides a pharmaceutical composition comprising (a) a pharmaceutically effective amount of Bambusae Caulis in Liquamen extract; And (b) provides a pharmaceutical composition for the treatment or prevention of diseases caused by oxidative damage (free radical) by a free radical comprising a pharmaceutically acceptable carrier.

The present inventors have tried to extract the active ingredient having an improved anti-inflammatory and antioxidant activity among various natural products, confirming that the Bambusae Caulis in Liquamen extract has anti-inflammatory and antioxidant activity, and active from the extract Compounds 1, 2-dihydroxybenzene and 1,4-dihydroxybenzene separated by activity guided isolation are nitric oxides. The present invention was completed by experimentally confirming that the formation inhibitory activity and the free radical scavenging ability have very excellent anti-inflammatory and antioxidant activity.

As used herein, the term "Bambusae Caulis in Liquamen" refers to a distillate obtained by heating bamboo at a high temperature, and in particular, in Korea, a node of rice pad (Phllostachys nigra Munro var.henosis Stapf) It also refers to the juice that leaks when burning the stem.

As used herein, the term "thrust extract" refers to a mixture obtained by extraction using a suitable solvent from the above forces.

The method of separating the bamboo extract, which is an active ingredient in the composition of the present invention, is carried out according to a conventional method known in the art for extracting the extract from natural products, that is, using a conventional solvent under the presence of conventional temperature and pressure. can do.

As an extraction solvent for extracting the bamboo extract of the present invention, a solvent which can be generally used may be used, and water, anhydrous or hydrous lower alcohol having 1 to 4 carbon atoms, acetone, ethyl acetate, butyl acetate and 1,3-butyl It is preferably extracted using a solvent selected from the group consisting of ene glycols, more preferably ether.

According to a preferred embodiment of the present invention, the torsion extract in the composition of the present invention is an ether solvent extract of torsion.

According to another aspect of the present invention, the present invention provides a pharmaceutical effective amount of (a) 1, 2-dihydroxybenzene (1,2-dihydroxybenzene) or 1, 4-dihydroxybenzene (1,4-dihydroxybenzene) ; And (b) provides a pharmaceutical composition for the treatment or prevention of inflammatory diseases comprising a pharmaceutically acceptable carrier.

According to another aspect of the present invention, the present invention provides a pharmaceutically effective amount of (a) 1, 2-dihydroxybenzene (1,2-dihydroxybenzene) or 1, 4-dihydroxybenzene (1,4-dihydroxybenzene) ; And (b) provides a pharmaceutical composition for the treatment or prevention of diseases caused by oxidative damage by free radicals comprising a pharmaceutically acceptable carrier.

The present inventors conducted activity guided fractionation and isolation to identify anti-inflammatory and antioxidant actives from the extracts of pulverulent extracts, which are phenolic compounds 1,2-dihydroxybenzene and 1 It was confirmed that 1,4-dihydroxybenzene has anti-inflammatory activity that inhibits the production of nitric oxide (NO) and antioxidant activity that eliminates free radicals.

The phenolic compounds 1,2-dihydroxybenzene and 1,4-dihydroxybenzene, which are the active ingredients of the composition of the present invention, are separated from the above-mentioned extracts by performing a conventional purification process. Separation via various purification methods additionally carried out, for example, separation using an ultrafiltration membrane having a constant molecular weight cut-off value, separation by various chromatography (manufactured for separation according to size, charge, hydrophobicity or affinity). do.

Inflammatory diseases that can be treated or prevented by the compositions of the present invention include sinusitis, rhinitis, conjunctivitis, asthma, dermatitis, atopic dermatitis dermatitis, inflammatory bowel disease, inflammatory collagen vascular diseases, glomerulonephritis, inflammatory skin diseases, and sarcoidosis, and the like. .

Diseases caused by oxidative damage by free radicals that can be treated or prevented by the compositions of the present invention include atherosclerosis, cancer, emphysema, cirrhosis and arthritis. Including but not limited to.

The pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier in addition to the active ingredient, the extract of the extract, the compound 1,2-dihydroxybenzene or 1,4-dihydroxybenzene. Pharmaceutically acceptable carriers included in the pharmaceutical compositions of the present invention are those commonly used in the preparation, such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, Calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like It doesn't happen. The pharmaceutical composition of the present invention may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives and the like in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

The appropriate dosage of the pharmaceutical composition of the present invention may vary depending on factors such as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate, . On the other hand, the oral dosage of the pharmaceutical composition of the present invention is preferably 0.001-1000 mg / kg (body weight) per day.

The pharmaceutical composition of the present invention may be administered orally or parenterally, and when administered parenterally, may be administered by intravenous infusion, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, or the like. In the pharmaceutical composition of the present invention, the route of administration is preferably determined depending on the type of disease to which it is applied.

The concentration of the extract, the compound 1,2-dihydroxybenzene or 1,4-dihydroxybenzene, which is an active ingredient included in the composition of the present invention, may be determined in consideration of the purpose of treatment, the condition of the patient, the period of time required, and the like. It is not limited to a specific range of concentrations.

The pharmaceutical composition of the present invention may be formulated into a unit dose form by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by a person having ordinary skill in the art to which the present invention belongs. Or by intrusion into a multi-dose container. The formulations may be in the form of solutions, suspensions or emulsions in oils or aqueous media, or in the form of excipients, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents.

According to another aspect of the present invention, the present invention provides a functional food composition for improving or alleviating an inflammatory disease comprising a bamboo extract (Bambusae Caulis in Liquamen) as an active ingredient.

According to another aspect of the present invention, the present invention provides a functional food composition for ameliorating or alleviating an inflammatory disease comprising 1, 2-dihydroxybenzene or 1, 4-dihydroxybenzene as an active ingredient.

According to another aspect of the present invention, the present invention provides a functional food composition for amelioration or alleviation of a disease caused by oxidative damage by free radicals including Bambusae Caulis in Liquamen extract as an active ingredient.

According to another aspect of the present invention, the present invention is for the improvement or alleviation of diseases caused by oxidative damage by free radicals containing 1, 2-dihydroxybenzene or 1, 4-dihydroxybenzene as an active ingredient Provides a functional food composition.

The force extract of the present invention or the active compound isolated therefrom can be used as an active ingredient in functional food compositions for improving or alleviating diseases caused by inflammatory diseases or oxidative damage by free radicals.

The functional food composition of the present invention includes components that are ordinarily added during the manufacture of food, and includes, for example, proteins, carbohydrates, fats, nutrients, and seasonings. For example, when prepared with a drink, flavoring agents or natural carbohydrates may be included as additional ingredients in addition to the extract of hawthorn as an active ingredient. For example, natural carbohydrates include monosaccharides (eg, glucose, fructose, etc.); Disaccharides (eg maltose, sucrose, etc.); oligosaccharide; Polysaccharides (eg, dextrins, cyclodextrins, etc.); And sugar alcohols (eg, xylitol, sorbitol, erythritol, and the like). As the flavoring agent, natural flavoring agents (e.g., taumartin, stevia extract, etc.) and synthetic flavoring agents (e.g., saccharin, aspartame, etc.) can be used.

The bamboo extract of the present invention and the active compound 1,2-dihydroxybenzene (1,2-dihydroxybenzene) or 1,4-dihydroxybenzene (1,4-dihydroxybenzene) isolated therefrom are nitrogen oxides (NO, Nitric) It has the ability to inhibit the production of oxides and has the scavenging ability of free radicals, so it can be developed as an anti-inflammatory drug and an antioxidant.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Experimental Method

1. Preparation of Death

Bamboo purchased 20L in July 2006 from Bamboo Bio Tech (Damyang, Jeollanam-do, Korea).

2. Fraction and Separation of Force

20 L of mortality was repeatedly extracted with ether at room temperature and concentrated under reduced pressure to obtain about 386.18 g of mortar extract. A single compound component was separated by liquid chromatography from the obtained ether extract. 26.78 g of ether extracts were prepared using a silica gel 60 column: 0.040-0.063 mm, 400 g, 10 x 100 cm; Merck KGaA, Darmstadt, Germany, hexan: chlorform: methanol = 13: 2: 1 (volume ratio) Separation was carried out under the solvent condition of to obtain three fractions of 6.02, 1.23, and 0.47 g. Repeat column chromatography on silica gel column (Silica gel 60 column: 0.040-0.063 mm, 100 g, 5 x 60 cm) for the first fraction using hexan: ethylacetate = 7: 1 and chloroform: methanol = 80: 1 143 mg of 2, 6-dimethoxyphenol (syringol) as a compound 1 was obtained by carrying out. Concentrate sequentially from 30% methanol to 100% methanol using MCI-gel CHP 20P (0.075-0.150 mm, 100 g, 5 X 60 cm; Mitsubishi, Tokyo, Japan) for the second fraction Compound 2 (compound 2), 1,2-dihydroxybenzene (1,2-dihydroxybenzene, pyrocatechol) 14 mg was isolated. For the third fraction, MCI-gel CHP 20P was used to sequentially increase the concentration from water to 100% methanol (MeOH). Compound 3 (compound 3), 1,4-dihydroxybenzene (1,4-dihydroxybenzene) 19 mg of hydroquinone and 125 mg of 1,2-dibenzenedicarboxylate (phthalate) as compound 4 were isolated. The above processes were carried out through monitoring by thin layer chromatography (TLC), and the plate used was silica gels 60 F ₂₅₄ plate (Merck KGaA, Darmstadt, Germany). After spraying FeCl ₃ or sprinkled with 10% H ₂ SO _{4 It} was confirmed by the method of coloring or by ultraviolet rays of 254nm (see Fig. 2).

3. Anti-inflammatory activity measurement

3-1. Cell culture

Mouse RAW 264.7 macrophage cells were purchased from Korean Cell Line Bank. Mouse RAW 264.7 macrophages were treated with DMEM medium (Sigma, St. Louis, MO, 10%) containing 10% FBS (fetal bovine serum), penicillin G (100 IU / ml), streptomycin (100 ug / ml). USA) was incubated in an incubator while maintaining a temperature of 37 ℃, 5% CO ₂ . The number of cells was counted using a hemocytometer.

3-2. Cytotoxicity measurement

In this experiment, MTT assay was used to determine the treatment concentration of each sample for mouse RAW 264.7 macrophages. This quantitative method is a modification of Mosmann's method ^{27 )} , which is performed by mitochondria dehydrogenase of living cells, MTT (3- (4,5-dimethylthiazol-2-yl) -2, a yellow soluble substrate. 5-diphenyl-tetrazolium bromide) (Sigma, St. Louis, Mo., USA) is used to reduce the dark blue water-insoluble formazan (formazan).

180 μl of mouse RAW 264.7 macrophages cultured in DMEM medium were added to the 96 wells with the culture solution and incubated for 2 hours to attach the cells, followed by 20 μl of the sample, followed by incubation for 24 hours. After incubation, the medium was removed, and 200 µl of MTT (5 mg / ml in PBS) prepared in advance was added and cultured again for 4 hours. After 4 hours, the medium was removed, and the dark blue crystals formed at this time were dissolved in 200 µl of DMSO to quantify the amount of formazan produced in purple. Since the amount of formazan produced is proportional to the number of living cells, the viability of the cells can be measured.

3-3. Measurement of NO Production Inhibitory Activity by iNOS Induced by LPS

This activity is measured by expressing nitric oxide synthase enzyme in mouse RAW 264.7 cells using lipopolysaccharide (LPS) and measuring the amount of nitric oxide (NO) produced by Griess's method ^29-30) . . Griess reagent (1% sulfanilamine, 0.1% N- (1-naphthyl) -ethylene diamine dihydrochloride, 2.5% H ₃ PO ₄ ) (Sigma, St. Louis, MO, USA) oxidizes NO to NO ₂ The produced NO ₂ was measured for absorbance at 540 nm and its concentration was determined using a calibration curve of NaNO ₂ .

180 μl of RAW 264.7 cells in 96 wells with medium, incubated for 2 hours to allow cells to adhere, 20 μl of LPS (1 μg / ml) and L-NMMA (NO synthesis inhibitory agent), a positive control 20 μl of each sample was added thereto and incubated for 24 hours, and the amount of NO generated in the culture solution was quantified using the Griess reaction reagent.

4. Antioxidant Activity Measurement

4-1. Free radical scavenging activity using DPPH

Antioxidant activity was determined based on the scavenging activity of stable DPPH free radicals (Sigma, St. Louis, MO, USA). Hatano's method ^{26 )} was applied, and 180 µl of 0.1 mM DPPH solution (99.5% ethanol) was added to 20 µl of the sample prepared for each concentration. After shaking for 10 seconds, the cells were incubated at 37 ° C. for 30 minutes, and then absorbance was measured at 492 nm using an ELISA reader (BIO-RAD, USA). L-ascorbic acid was used as a positive control drug.

5. Statistical Analysis

All data values are expressed as the mean value standard deviation of three or more experimental values. Each value was analyzed by SPSS using the Student-Newman-Keuls (S-N-K) test. At this time, the significance was determined based on p <0.05.

Experiment result

1. Check the structure

1-1. Compound 1

Compound 1 was a yellow amorphous powder. Spots were detected by UV irradiation at 254 nm. Brown spots were detected by spraying with ethanol FeCl ₃ , and light brown spots were detected by 10% H ₂ SO ₄ (heating). NMR measurement data of Compound 1 was as follows.

¹ H-NMR (300MHz, MeOH-d4): δ 3.80 (6H, s, 2 X OCH ₃ ), 6.62 (2H, d, J = 7.2 Hz, H-3, 5), 6.74 (1H, d, J = 7.2 Hz, H-4)

¹³ C-NMR (75 MHz, MeOH-d4): δ 56.6 (2 X OCH ₃ ), 106.7 (C-3, 5), 119.4 (C-4), 137.3 (C-1), 149.2 (C-2, 6)

EI-MS: m / z 154 [M] &lt; + &gt;

Compound 1 is described in Document ³⁴⁾ . It was identified as 2,6-dimethoxyphenol (Syringol) compared to the spectroscopic data of the reported values.

1-2. Compound 2

Compound 2 was in the form of a blue black amorphous powder. Spots were detected by UV irradiation at 254 nm. Blue-black spots were detected by spraying with ethanol FeCl ₃ , and light blue spots were detected by 10% H ₂ SO ₄ (heat). NMR measurement data of Compound 2 was as follows.

¹ H-NMR (300MHz, MeOH-d4): δ 6.64-6.80 (2H in total, m, H-3, 4), 6.73-6.77 (2H in total, m, H-2, 5)

¹³ C-NMR (75 MHz, DMSO-d6): δ 116.6 (C-4, 5), 121.2 (C-3, 6), 146.6 (C-1, 2)

EI-MS: m / z 110 [M] +

Compound 2 is described in Document ³⁴⁾ . It was identified as 1, 2-dihydroxybenzene (Pyrocatechol) compared to the spectroscopic data of the reported value.

1-3. Compound 3

Compound 3 was a white amorphous powder. Spots were detected by UV lamp irradiation at 254 nm. Gray spots were detected by spraying with ethanol FeCl ₃ , and light brown spots were detected by 10% H ₂ SO ₄ (heating). NMR measurement data of Compound 3 was as follows.

¹ H-NMR (300MHz, MeOH-d4): δ 6.64 (4H in total, s, H-2, 3, 5, 6)

¹³ C-NMR (75MHz, MeOH-d4): δ 116.4 (C-2, 3, 5, 6), 150.9 (C-1, 4)

EI-MS: m / z 110 [M] +

Compound 3 is ^described in It was confirmed as 1, 4-dihydroxybenzene (Hyquinone) compared with the spectroscopic data of the reported value.

1-4. Compound 4

Compound 4 was a black amorphous powder. Spots were detected under UV lamp irradiation at 254 nm. Blue-black spots were detected by spraying with ethanol FeCl ₃ and light blue spots were detected by 10% H ₂ SO ₄ (heating). NMR measurement data of Compound 4 was as follows.

¹ H-NMR (300MHz, DMSO-d6): δ 6.56-6.60 (2H in total, m, H-3, 4), 6.71-6.75 (2H in total, m, H-2, 5)

¹³ C-NMR (75 MHz, DMSO-d6): δ 127.6 (C-3, 4), 129.3 (C-2, 5), 133.2 (C-1, 6), 167.5 (-2COOH)

EI-MS: m / z 166 [M] &lt; + &gt;

Compound 4 is described in Document ³⁶⁾ . It was identified as 1, 2-benzene dicarboxylate (Phthalate) compared to the spectroscopic data of the reported value.

The structure of the compound 1-4 of this invention is as follows.

2. Anti-inflammatory activity measurement result

2-1. Cytotoxicity Measurement Results

Prior to the anti-inflammatory activity experiment using RAW 264.7 macrophage cells, MTT assay was performed to determine the toxicity of each sample to the cells and the concentration of sample processing. It can be seen that each fraction 1-3 of the extract of bamboo and each compound 1-4 isolated from the extract was not toxic at the treatment concentration of 12.5-100 uM (see FIGS. 3 and 4).

2-2. Measurement result of NO production inhibitory activity by iNOS induced by LPS

Inhibitory activity against NO production was determined by treating RAW 264.7 macrophages with each fraction and compound. LPS treatment significantly increased NO content. As concentration increased, fraction 1 showed little inhibitory activity on NO production, but fraction 2 (IC ₅₀ = 76.16 ± 5.07 ug / ml) and fraction 3 (IC ₅₀ = 67.43 ± 2.51 ug / ml) were positive control L- Compared with NMMA (IC ₅₀ <12.5 ug / ml) showed NO production inhibitory activity (p <0.05).

In addition, compound 2 (IC ₅₀ = 52.74 ± 2.63) and compound 3 (IC ₅₀ = 59.46 ± 1.02) showed significant NO production inhibitory activity compared to the positive control L-NMMA (IC ₅₀ <12.5 ug / ml) (p <0.05). However, Compound 1 and Compound 4 did not show inhibitory activity on NO production (see FIGS. 5 to 8).

[IC ₅₀ Values of Each Fraction and Compounds of Bamboo Extract on NO Production]

3. Measurement result of antioxidant activity

3-1. DPPH Freedom with Radical ( free radical ) Scatters  Measurement result

Scavenging activity of DPPH radicals was determined by treating each fraction and compound. As a result of measuring DPPH free radical scavenging ability, the fraction extract 2 (IC ₅₀ <12.5 ug / ml) and fraction 3 (IC ₅₀ <12.5 ug / ml) were found to be positive control ascorbic acid (IC ₅₀ ). <12.5 ug / ml) showed a similarly high degree of free radical scavenging activity. In addition, compound 2 (IC ₅₀ = 12.67 ± 0.44) and compound 3 (IC ₅₀ = 23.95 ± 3.82) showed similarly high levels of free radical scavenging activity compared to the positive control ascorbic acid (IC ₅₀ <12.5 ug / ml). (See FIGS. 9 and 10).

[IC ₅₀ Values of Each Fraction and Compounds of Bamboo Extracts on DPPH Radicals]

Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that the specific technology is merely a preferred embodiment, and the scope of the present invention is not limited thereto. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

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1 is a diagram showing the role of iNOS in the inflammatory response.

Figure 2 schematically shows the process of separating compounds 1-4 from the bamboo extract.

Figure 3 shows the results of measuring the effect of each fraction 1-3 of ether extract of kill on the viability of mouse RAW 264.7 macrophages. Cell viability was measured by MTT assay.

Figure 4 shows the results of measuring the effect of Compound 1-4 isolated from the kill extract on the viability of mouse RAW 264.7 macrophages. Cell viability was measured by MTT assay.

Figure 5 shows the NO content of each fraction 1-3 of the ether extract of P. aeruginus in LPS stimulated mouse RAW 264.7 macrophage cells.

FIG. 6 shows inhibitory activity against NO production when LPS-stimulated mouse RAW 264.7 macrophage cells were treated with fractions 1-3 of ether extracts.

FIG. 7 shows NO content when treated with Compound 1-4 isolated from P. aeruginosa extract in LPS stimulated mouse RAW 264.7 macrophage cells.

FIG. 8 shows inhibitory activity against NO production when treated with Compound 1-4 isolated from P. aeruginosa extract in LPS stimulated mouse RAW 264.7 macrophage cells.

Figure 9 shows the results of measuring the DPPH radical scavenging activity of each fraction 1-3 of the ether extract.

Figure 10 shows the results of measuring the DPPH radical scavenging activity of Compound 1-4 isolated from the bamboo extract.

Claims (10)

(a) a pharmaceutically effective amount of an ether extract of Bambusae Caulis in Liquamen; And (b) a pharmaceutical composition for treating or preventing conjunctivitis, asthma, or inflammatory bowel disease comprising a pharmaceutically acceptable carrier. delete delete Functional food composition for improving or alleviating conjunctivitis, asthma, or inflammatory bowel disease, comprising an ether extract of Bambusae Caulis in Liquamen as an active ingredient. delete (a) a pharmaceutically effective amount of an ether extract of Bambusae Caulis in Liquamen; And (b) a pharmaceutical composition for the treatment or prophylaxis of atherosclerosis, cirrhosis or arthritis, comprising a pharmaceutically acceptable carrier. delete delete Functional food composition for improving or alleviating atherosclerosis, cirrhosis or arthritis, comprising ether extract of Bambusae Caulis in Liquamen as an active ingredient. delete

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