KR101556773B1 - Anti-inflammatory composition containing diterpenes from the roots of oriza sativa l. - Google Patents

Abstract

The present invention relates to a composition for preventing, ameliorating or treating inflammation comprising a diterpene compound.

Description

FIELD OF THE INVENTION [0001] The present invention relates to an antiinflammatory composition comprising a diaperpen compound derived from a rice plant root,

The present invention is directed to anti-inflammatory compositions.

Rice ( Oryzasativa L. ) is a perennial herbaceous plant belonging to the Gramineae family, and is widely distributed throughout Asia. Height is 50 to 100 cm. It grows by forming branches in the branches near the roots. When flowers bloom, they stand upright, but when they are ripe, they fall downward. Many minorities run. Flowers bloom around July to August, and they are eliminated by their own moisture.

Rice, which is the fruit of rice, is the main food resource of Korea. During that time, methods of processing rice, rice, improvement methods (for example, Korean Patent Publication No. 10-2013-0141261), foods using rice or rice by- Korean Patent Laid-Open No. 10-2013-0065061, etc.). However, it has recently been shown that rice has many useful ingredients.

Accordingly, the present inventors isolated new compounds from rice while isolating and studying the compounds derived from rice, and confirmed that the specific diterpene compounds containing them have anti-inflammatory activity, thus completing the present invention.

It is an object of the present invention to provide an anti-inflammatory composition.

In order to achieve the above object, the present invention provides an anti-inflammatory composition comprising a diterpene compound derived from a root of rice and an acceptable salt thereof.

The anti-inflammatory composition of the present invention has the ability to inhibit nitric oxide production.

Figure 1 shows the inhibitory effect of the rice root extract and fractions of Examples 1 to 5 on nitric oxide production.
Figure 2 shows the cell viability of the rice root extract and fractions of Examples 1-5.
Fig. 3 shows the ability of the compounds 1 to 5, which are diether compounds derived from rice roots, to produce nitric oxide.
Figure 4 shows the cell viability of compounds 1 to 5, which are dietherpenic compounds isolated from rice roots.
FIG. 5 shows the ability of compounds 1 to 5 to inhibit the expression of mRNA and protein of iNOS (A: reverse transcriptase-polymerase chain reaction analysis, B: Western blot analysis).

The present invention

The present invention relates to a pharmaceutical composition for preventing or treating an inflammatory disease comprising a compound represented by the following formula (1) or an acceptable salt thereof.

≪ Formula 1 >

Wherein R 1 is H or OR 2, and R ₂ is selected from the group consisting of H, CH ₃ , CH ₂ CH ₃ and CH ₂ CH ₂ CH ₃ .

(2)

(3)

≪ Formula 4 >

In this case, R3 is H or OR4, wherein R4 is is one which is selected from the group consisting of _{H, CH 3, CH 2 CH} 3 and CH ₂ CH ₂ CH _3.

Further, according to the present invention,

The present invention relates to a food composition for preventing or ameliorating inflammation, which comprises a compound represented by the following formula (1) or an acceptable salt thereof.

≪ Formula 1 >

Wherein R 1 is H or OR 2, and R ₂ is selected from the group consisting of H, CH ₃ , CH ₂ CH ₃ and CH ₂ CH ₂ CH ₃ .

(2)

(3)

≪ Formula 4 >

In this case, R3 is H or OR4, wherein R4 is is one which is selected from the group consisting of _{H, CH 3, CH 2 CH} 3 and CH ₂ CH ₂ CH _3.

Further, according to the present invention,

The present invention relates to a cosmetic composition for preventing or improving inflammation, which comprises a compound represented by the following formula (1) or an acceptable salt thereof.

≪ Formula 1 >

Wherein R 1 is H or OR 2, and R ₂ is selected from the group consisting of H, CH ₃ , CH ₂ CH ₃ and CH ₂ CH ₂ CH ₃ .

(2)

(3)

≪ Formula 4 >

In this case, R3 is H or OR4, wherein R4 is is one which is selected from the group consisting of _{H, CH 3, CH 2 CH} 3 and CH ₂ CH ₂ CH _3.

Also,

The present invention relates to a compound represented by the following formula (1) or an acceptable salt thereof.

≪ Formula 1 >

Wherein R 1 is OR 2 and R ₂ is selected from the group consisting of H, CH ₃ , CH ₂ CH ₃ and CH ₂ CH ₂ CH ₃ .

Also,

The present invention relates to a compound represented by the following formula (4) or an acceptable salt thereof.

≪ Formula 4 >

In this case, R3 is H or OR4, wherein R4 is is one which is selected from the group consisting of _{H, CH 3, CH 2 CH} 3 and CH ₂ CH ₂ CH _3.

The present invention also relates to a process for producing the compound or a salt thereof, which comprises extracting rice with an organic solvent.

Hereinafter, the present invention will be described in detail.

Pharmaceutical composition

The present invention relates to a pharmaceutical composition for the prophylaxis or treatment of inflammatory diseases comprising a compound of the present invention or a pharmaceutically acceptable salt thereof. The pharmaceutical composition has a property of inhibiting the expression of nitric oxide (NO).

The pharmaceutical composition of the present invention may contain 0.01 to 80% by weight, preferably 0.02 to 65% by weight, of the compound of the present invention or a pharmaceutically acceptable salt thereof. However, this can be increased or decreased according to the need of the medicinal person, and it can be appropriately increased or decreased according to the situation such as the diet, nutritional status, disease progression, degree of inflammation.

The pharmaceutical composition of the present invention can be administered orally or parenterally and can be used in the form of a general pharmaceutical preparation. The preferred pharmaceutical preparations are those for oral administration such as tablets, hard or soft capsules, liquids, suspensions, etc. These pharmaceutical preparations can be prepared in conventional pharmaceutically acceptable carriers, for example, as excipients for oral preparations, Binders, disintegrators, lubricants, solubilizers, suspending agents, preservatives or extenders.

The dosage of the pharmaceutical composition comprising the compound of the present invention or a pharmaceutically acceptable salt thereof of the present invention can be determined by an expert according to various factors such as the condition of the patient, age, sex, and complications, May be administered at a dose of 0.1 mg to 10 g, preferably 10 mg to 5 g, per kg of the adult. Also, the daily dosage of the pharmaceutical composition per unit dosage form, or a half, 1/3 or 1/4 dose thereof, may be contained, and may be administered 1 to 6 times per day. However, in the case of long-term intake for the purpose of health and hygiene or for the purpose of controlling health, the amount may be less than the above range, and the active ingredient may be used in an amount of more than the above range since there is no problem in terms of safety.

Inflammatory disease

The inflammatory disease is selected from the group consisting of edema, dermatitis, allergy, atopy, asthma, conjunctivitis, periodontitis, rhinitis, otitis, sore throat, tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn's disease, colitis, hemorrhoids, May be any one selected from the group consisting of fibromyalgia, psoriatic arthritis, osteoarthritis, rheumatoid arthritis, shoulder periarthritis, tendonitis, nephritis, myositis, hepatitis, cystitis, nephritis and multiple sclerosis.

The Diterpen  Compounds

The compounds of the formulas 1 to 4 of the present invention, particularly the compounds of the compounds 1 to 5, have the ability to prevent, improve or treat inflammation. The diaperpen compounds of the present invention can be isolated from plants or obtained through synthesis. The plant is preferably a paddy field plant, more preferably rice.

The Diterpen  Methods for the preparation of compounds

The present invention provides a process for producing the dietherpenic compounds of the present invention comprising extracting rice with an organic solvent. After the extraction with the organic solvent, the organic solvent may be further fractionated with an organic solvent which is the same as or different from the organic solvent. The organic solvent of the present invention may be a low-cost alcohol having 5 or less carbon atoms, ethyl acetate, water, chloroform, etc., and the lower alcohol is preferably methanol, ethanol, propanol or butanol.

Food composition

The present invention is directed to a food composition for preventing or ameliorating an inflammatory disease comprising a compound of the present invention or an acceptable salt thereof.

The food of the present invention may be, but is not limited to, a health supplement food, a health functional food, a functional food, etc., and includes the addition of a compound of the present invention or an acceptable salt thereof to natural foods, processed foods, .

The food composition of the present invention can be used appropriately as it is, or can be used in combination with other food or food compositions, or can be used appropriately according to conventional methods. The amount of the active ingredient to be mixed can be suitably determined according to its intended use (prevention, improvement, or therapeutic treatment). In general, the compound of the present invention or an acceptable salt thereof may be added in an amount of 0.1 to 70% by weight, preferably 2 to 50% by weight, based on 100% by weight of the raw material of the food or beverage in the production of food or beverage . The effective dose of the compound of the present invention or an acceptable salt thereof may be used in accordance with the effective dose of the pharmaceutical composition but may be less than the above range for the purpose of health and hygiene or for long- , And the active ingredient can be used in an amount in the above range because there is no problem in terms of safety.

There is no particular limitation on the kind of the food. The food composition may be used in the form of tablets, hard or soft capsules, liquids, suspensions, and the like, which may contain conventional excipients, such as excipients in the case of oral preparations, Binders, disintegrators, lubricants, solubilizers, suspending agents, preservatives or extenders.

Examples of foods to which the compounds of the present invention or acceptable salts thereof can be added include dairy products including meats, sausages, breads, chocolates, candies, snacks, confectionery, pizza, ramen noodles, other noodles, gums, ice cream, , Beverages, tea, drinks, alcoholic beverages and vitamin complexes, and other nutrients, but are not limited to these kinds of foods.

Cosmetics  Composition

The present invention is directed to a cosmetic composition comprising a compound of the invention or an acceptable salt thereof. The cosmetic composition of the present invention is effective for anti-inflammation, and can be used especially for troublesome skin, acne skin, atopic skin and the like. The cosmetic composition of the present invention may be prepared in any formulations conventionally produced in the art and may be formulated into a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant- containing cleansing oil, powder foundation , An emulsion foundation, a wax foundation and a spray, but is not limited thereto. The cosmetic composition of the present invention may also be formulated as a softening agent, a nutritional lotion, a nutritional cream, a massage cream, an essence, an eye cream, a cleansing cream, a cleansing foam, a cleansing water, a pack, a spray or a powder.

The cosmetic composition of the present invention may contain a skin penetration promoter for effective transdermal absorption. The skin penetration promoting agent is not particularly limited so long as it is a general skin penetration promoting agent used in the cosmetic composition in the art. For example, the skin penetration promoter of the present invention may be a fatty acid / ester compound such as dimethylsulfoxide, oleic acid, limonene, and the like.

When the cosmetic composition of the present invention is used, transdermal absorption may be promoted by using physical methods such as shock waves, iontophoresis, electrical invasion, and microinfiltration.

Feed composition

The present invention is directed to a feed composition comprising a compound of the invention or an acceptable salt thereof. The feed composition of the present invention can be seeded with conventional feeds, and the feed composition of the present invention can be added to a conventional feed composition to prepare a functional feed composition. In addition, the feed composition of the present invention may further comprise a functional ingredient in addition to the compound of the present invention or an acceptable salt thereof. In the preparation of a functional feed composition in which the conventional feed composition and the compound of the present invention or an acceptable salt thereof are mixed, the compound of the present invention or an acceptable salt thereof is added in an amount of 0.01 to 30.00% by weight, preferably 0.01 By weight to 20.00% by weight. The effective dose of the compound of the present invention or the acceptable salt thereof in the feed composition can be used in accordance with the effective dose of the food composition but may be less than the above range in the case of long-term intake for continuous health control, There is no problem in terms of safety, so that it can be used in an amount exceeding the above range as necessary.

The feed composition of the present invention is intended for livestock or poultry. The livestock or poultry may be any kind of animal such as cattle, pigs, chickens, horses, sheep, donkeys, mules, boars, rabbits, quail, ducks, fowls, poultry chickens, pigeons, turkeys, dogs, cats, monkeys, hamsters, , Parrots, parakeets, canaries, and the like, but are not limited to mammals or birds other than humans capable of breeding in the home.

BRIEF DESCRIPTION OF THE DRAWINGS The advantages and features of the present invention and the manner of achieving them will become apparent with reference to the embodiments described in detail below with reference to the accompanying drawings. It should be understood, however, that the invention is not limited to the disclosed embodiments, but is capable of many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, To fully disclose the scope of the invention to those skilled in the art, and the invention is only defined by the scope of the claims.

Materials and methods

Roots of rice

Rice (Chucheongbyeo) roots were collected in November 2011 in Ganghwa-gun, Incheon city, and Dr. Cho Jun-hyun of Rural Development Administration identified them.

Tools & Reagents

The silica gel (SiO2) for the column chromatography was Kieselgel 60 (Merck, Darmstadt, Germany), octadecyl SiO2 (ODS) was used for LiChroprep RP-18 (40-60 μm, Merck), Sephadex LH-20 (Amersham Pharmacia Biotech, Uppsala, Sweden) ) Were used. TLC was performed with Kieselgel 60 F254 and RP-18 F254S, and UV detection using a UV lamp (Spectroline, Model ENF-240 C / F, Spectronics Corporation, New York, USA) H2SO4 was sprayed and heated to investigate the color development. NMR spectra were measured using a Varian Inova AS 400 (400 MHz, Varian, Palo Alto, CA, USA) and Circular dichroism (CD) using Chirascan Plus (Applied Photophysics Ltd., Leatherhead, UK). The IR spectra were measured using a Perkin model 599B (Perkin-Elmer, Waltham, Mass., USA) and EI-MS was measured using JMS-700 (JEOL, Tokyo, Japan). The matrix was glycerol Were used. Melting points were measured using a Stanford Research System melting point meter (SRS, Sunnyvale, Calif., USA) and the non-linearity was measured using Polarimeter P-1020 (JASCO, Tokyo, Japan).

Cell culture

The murine macrophage RAW264.7 was purchased from the Korean Cell Line Bank (KCLB), and 10% fetal bovine serum (FBS, WelGENE Inc., Seoul, Korea), glutamate (2 mM), penicillin The cells were cultured in a Dulbecco's modified Eagle's medium (DMEM, WelGENE) medium containing 5% CO ₂ at 37 ° C with streptomycin (100 μg / mL). Samples containing various concentrations of rice root extract, fractions and diterpene compounds were treated with lipopolysaccharide (LPS, Sigma-Aldrich Co.) (1 μg / mL) or with LPS alone and RAW 264.7 cells were treated with 18-24 Lt; / RTI >

Nitrogen oxide measurement

A frequency divider so that the RAW264.7 cells per well of 96-well cell culture plate in 1 × 10 ⁴ cells were then incubated for 12 to 18 hours while attached. Various concentrations of rice root extract, fractions and diterpene compounds were treated simultaneously with LPS (1 [mu] g / ml) or LPS alone for 24 hours. After 24 hours, the cell culture was obtained and the amount of NO contained in the culture was measured using a Griess Reagent System (Sigma-Aldrich Co.). 100 占 퐇 of the cell culture medium and 100 占 퐇 of the Griess reagent in a new 96-well plate were mixed and allowed to stand at room temperature for 10 minutes. The absorbance was measured at 550 nm using a microplate reader (Bio-Tek Instruments, Inc., Wilnooski, VT, USA). The amount of nitric oxide produced was calculated using sodium nitrite standard curve.

Cytotoxicity test

A frequency divider so that the RAW264.7 cells per well of 96-well cell culture plate in 1 × 10 ⁴ cells were then incubated for 12 to 18 hours while attached. Various concentrations of rice root extract, fractions and diterpene compounds were dissolved in serum-free medium and treated for 24 hours. After 24 hours, 10 [mu] l of a solution of 10 [mu] g / mL of 3- (4,5-dimetnythiazolyl-2) -2,5-diphenyl-thetazoliumbromide (MTT, Sigma-Aldrich Co.) was further added thereto. After 30-60 minutes, the cell culture solution was completely removed and the purple-colored formazan crystals were completely dissolved in 100 μl of dimethyl sulfoxide (DMSO, Sigma-Aldrich Co.) for each well. Absorbance was measured. The survival rate of the cells was determined by the ratio of survival cells in the control group treated with the sample according to the following formula 1 and the control group without any treatment.

<Formula 1>

Cell viability (%) = [OD (absorbance of sample group) - OD (absorbance of blank group) / OD (control absorbance) - OD (absorbance of blank group)

Reverse transcriptase - Polymerase chain reaction  analysis

RAW264.7 cells were seeded on a 6-well cell culture plate at a density of 1 × 10 ⁵ cells / well and cultured for 12 to 18 hours while adhering. Diterpen compounds were treated simultaneously with LPS (1 [mu] g / ml) or LPS alone for 18 hours in RAW264.7 cells. After 18 hours, total RNA of the cells was extracted using a Trizol reagent kit (Invitrogen, Carsbad, CA, USA) and 5 μg of RNA was reverse-transcribed using M-MuLV reverse transcription polymerase (Fermentas Life Science) Respectively. cDNA encoding iNOS and GAPDH was amplified by polymerase chain reaction (PCR) using Taq polymerase using a forward primer and a reverse primer, respectively. The primers used for amplification are shown in Table 1 below, and GAPDH was used as an internal control.

Western blot  analysis

RAW264.7 cells were seeded on a 6-well cell culture plate at a density of 1 × 10 ⁵ cells / well and cultured for 12 to 18 hours while adhering. Diterpen compounds were treated simultaneously with LPS (1 [mu] g / ml) or LPS alone for 18 hours in RAW264.7 cells. After 18 hours, the cells were lysed using a RIPA buffer solution (Biosesang Inc., Seoul, Korea) containing a protease inhibitor and the intracellular proteins were recovered. The recovered protein was quantified using a Bradford (Biosesang Inc.) protein assay kit and SDS-polyacrylamide gel electrophoresis was performed on 8% gel using 40 μg of the quantified protein. After electrophoresis, the proteins separated by nitrocellulose membrane were transferred and membrane was incubated with Tris-buffered saline-Tween (TBS-T) containing 5% skim milk for 1 hour at room temperature to inhibit non-specific binding of antibody Respectively. After incubation for 12 hours with an antigen specific primary antibody, secondary antibody with horseradish peroxidase was reacted at room temperature for 2 hours. After washing with TBS-T 6 times or more, it was confirmed by ECL detection system (Amersham Bioscience, Piscataway, NJ, USA). GAPDH was used as an internal control to confirm that the total amount of protein used in the experiment was the same.

Rice root extract and Fraction  Produce

8.35 kg of dried rice roots was extracted three times for 12 hours with 80% methanol (94 L), and the obtained filtrate was concentrated under reduced pressure to obtain 284 g (ORM, rice root methanol extract, Example 1).

The rice seed methanol extract of Example 1 was separated and extracted with ethyl acetate (EtOAc, 4 L × 3) / H ₂ O (4 L). The ethyl acetate layer was removed and the remaining water layer was washed with n-butanol butanol (n-BuOH, 2.8 L x 3). Each layer was concentrated under reduced pressure to obtain ethyl acetate fraction (ORE, Example 2, 38 g), n-butanol fraction (ORB, Example 3, 26 g) and water fraction (ORH, Example 4).

Isolation of Diterpenic Compounds from Rice Root Fractions

To 38 g of the ethyl acetate fraction (ie, Example 2) was added silica gel column chromatography (cc) (φ 11.5 × 13.0 cm, n-hexane-ethyl acetate = 7: 1, 8 L → 5: 3: 1, 17 L → 1: 1, 10 L → CHCl 3 -MeOH = 17: 1, 4 L → 15: 1, 7 L → 12: 2 L? 3: 1, 4 L? 1: 1, 6 L) to obtain 32 fractions (ORE-1 to ORE-32). Ore3 fraction (2 g, Ve / Vt = 0.01 - 0.02; volume of the fraction eluting solvent; volume of the total eluting solvent used for elution of Vt) silica gel cc (φ 5.0 × 14.0 cm, n-hexane-ethyl acetate = 27: 1, 2 L) to obtain 15 fractions (ORE-3-1 to ORE-3-15). ODS cc (φ3.0 × 7.0 cm, acetone-acetonitrile-H2O = 5: 5: 2, 1.2 L) was applied to the ORE-3-7 fraction (130 mg, Ve / Vt 0.06 to 0.11) 17 fractions (ORE-3-7-1 to ORE-3-7-17) were obtained. Compound 2 [ORE-3-7-8, 10.7 mg, Ve / Vt 0.16-0.37, TLC F254s) Rf 0.20, acetone-acetonitrile-H2O = 3: 3: 1]. ODS cc (φ 4 × 8 cm, acetone-acetonitrile-H 2 O = 1: 1: 4, 2.0 L → 1: 1: 2, 1.0 L) was added to the ORE-12 fraction (692 mg, Ve / Vt 0.19 to 0.21) 25 fractions (ORE-12-1 to ORE-12-25) were obtained. Sephadex LH-20 cc (φ1.5 × 56.0 cm, MeOH-H2O = 9: 1, 0.8 L) was applied to the ORE-12-8 fraction (100 mg, Ve / Vt 0.30 to 0.34) ORE-12-8-1 to ORE-12-8-8) and ORE-12-8-3 fraction (2 g, Ve / Vt = 0.43 to 0.48) in silica gel cc (? 3.0 X 12.0 cm , CHCl3-MeOH = 40: 1, 0.7 L) to obtain 13 fractions (ORE-12-8-3-1 to ORE-12-8-3-13). Silica gel cc (φ2.5 × 10.5 cm, n-hexane-ethyl acetate = 8: 1, 0.8 L) was applied to the ORE-12-8-3-4 fraction (32 mg, Ve / Vt = 0.15 to 0.25) 12 fractions (ORE-12-8-3-4-1 to ORE-12-8-3-4-12) were obtained, and Compound 5 [ORE-12-8-3-4-2, 2.7 mg, Ole-12-8-3-4-10, 4.9 mg, Ve / Vt 0.63 to (TLC (SiO2 F254) Rf 0.67, n-hexane-ethyl acetate = 3: 1) 0.89, TLC (SiO2 F254) Rf 0.23, n-hexane-ethyl acetate = 3: 1]. ODS cc (φ 4.5 X 12.0 cm, acetone-acetonitrile-H 2 O = 1: 1: 4, 2.0 L → 3: 3: 2, 1.2 L) was added to ORE-14 fraction (1.9 g, Ve / Vt 0.22-0.27) ORE-14-6 fractions (93 mg, Ve / Vt 0.49 to 0.58) were obtained from silica gel cc (φ3.0 X (ORE-14-6-1 to ORE-14-6-12) were obtained by subjecting compound 1 [ORE-14-6- 3, 35 mg, Ve / Vt 0.13 to 0.15, TLC (SiO2 F254) Rf 0.60, CHCl3-MeOH = 20: 1]. ODS cc (φ3.0 × 5.0 cm, acetone-acetonitrile-H2O = 2: 2: 7, 1.8 L) was applied to the ORE-20 fraction (596 mg, Ve / Vt 0.34-0.41) ORE-20-2 fraction (127 mg, Ve / Vt 0.05-0.07) was purified with silica gel cc (φ3.5 × 12.0 cm, CHCl3-MeOH = 35 ORE-20-2-8, 4.5 mg, Ve / Vt (1: 1.5 L) to obtain 17 fractions (ORE-20-2-1 to ORE-20-2-17) 0.24 to 0.33, TLC (SiO2 F254) Rf 0.29, CHCl3-MeOH = 19: 1].

Isolated Dieter Pen  Identification of compounds

NMR, MS, and IR were measured on the separated compounds to identify the structure. As a result, compounds 1, 2, and 4 were identified as previously known compounds, momilactone A, sandaracopimaradien-3-one, and oryzalexin A, respectively , Compounds 3 and 5 were identified as novel compounds.

Compound 1 confirmed the absorption of lactone (1770 cm ^-1 ), ketone (1700 cm ^-1 ), and double bond (1640 cm ^-1 ) in the IR spectrum. A 314 ion peak was identified in the EI / MS spectrum and the molecular weight was determined to be 314. (1H, dd, J = 17.6, 10.8 Hz, H-15), 4.92 (1H, dd, J = 17.6, 1.2 Hz, H-16a), and 4.87 (OH) 2), one oxygenated methine signal [δH 4.79 (1H, dd, J = 10.8, 1.2 Hz, H-16b) (1H, dd, J = 5.8, 5.2 Hz, H-6). In the high magnetic field region, three methyl signals [δH1.49 (3H, s, H-18), 1.25 (3H, s, H-20), and 0.83 A number of methine and methylene signals were observed around 2.54 to 1.27. In the 13C-NMR, 20 signals were observed and expected to be diterpenoids. Olefin metyelene [delta C 110.0 (C-16)]], olefin quaternary [delta C 147.9 (C-8)], olefin methyelene [delta C 110.0 oxygenated methine [δC 73.0 (C-6)] signals were observed and two olefin methines [δC 148.8 (C-15) and 113.8 (C-7)] were observed. In the high magnetic field region, two quaternary [δC 53.4 (C-4), 39.9 (C-13), and 32.3 (C-10)], two methines [δC 50.0 ], Five methylene [47.4 (C-14), 37.1 (C-12), 34.7 (C-2), 31.0 (C-1), and 23.8 C-17), 21.6 (C-20), and 21.3 (C-18)] signals were observed. From the above results, Compound 1 was determined to be a pimarane type diterpenoid containing one ketone, one lactone, and two double bonds.

The oxygenated methine proton signal of δ _H 4.79 (H-6) in gCOSY showed the proline signal of δ _H 5.64 (H-7) and the proline signal and cross peak of δH2.29 (H-5). In the gHMBC, the ketone carbon signal C-3 (δC 205.1) showed a cross peak with the methyl proton signal H-18 (δH 1.49) and methylene proton signal H-1 (δH 1.85), while the lactone C-19 (δC 174.2) -18, and methine proton signal H-5 (δH 2.29). H-18 showed cross peaks with the quaternary carbon signals C-4 (δC 53.4) and C-5 (δC 46.2) and the methyl proton signal H-20 (δH 1.25) with C-5 and methine carbon signal C -9 (δC 50.0) and cross peak. The methyl proton signal H-17 (δH 0.83) was the methylene carbon signal C-12 (δC 37.1), the olefin methine carbon signal C-15 (δC 148.8), and the methylene carbon signal C-14 (δC 47.4) C-18 (δC 21.3), and C-20 (δC 21.6)] of the methyl groups. H-16 (δH 4.92 and 4.87) H-17 (δH 5.64) showed a cross peak with C-6 (δC 73.0) and C-9, while H-17 and olefin methylene proton signals of H- carbon signal C-13 (δC 39.9) and a cross peak, indicating that two pairs of double bonds are located at C-7 and C-8, and at C-15 and C-16. From the above results, Compound 1 was identified as Momilactone A.

Compound 3 showed absorption of hydroxyl group (3374 cm ^-1 ), lactone (1767 cm ^-1 ), ketone (1699 cm ^-1 ) and double bond (1647 cm ^-1 ) in the IR spectrum and EI / MS spectrum The ion peak of 330 was observed and the molecular weight was determined to be 330. The molecular formula was determined as C20H26O4 by observing an ion peak of 330.1833 at high resolution EI / MS (calcd. 330.1832, for C20H26O4). The NMR data of Compound 3 was very similar to Compound 1, and another oxygenated quaternary carbon signal was observed at δC 74.4. The methyl proton signal H-18 (δH 1.64) in the gHMBC was the ketone carbon signal C-3 (δC 204.9), the quaternary carbon signal C-4 (δC 54.7), the methine carbon signal C-5 (δC 48.4) (Δ C 17.8) and lactone carbon signal C-19 (δ C 174.8) and the methyl proton signal H-20 (δH 1.32) were methylene carbon signals C-1 (δC 27.8), C-5, oxygenated quaternary carbon C-9 (δC 74.4) signal, and quaternary carbon signal C-10 (δC 38.0). Thus, compound 3 is a novel compound, 3-oxo-9beta-hydroxypimara-7,15-dien-19,6beta-olide -olide), and named as "momilactone D".

On the other hand, the absorption of hydroxyl (3620 cm ^-1 ), ketone (1705 cm ^-1 ), and double bonds (1614 cm ^-1 ) in the IR spectrum of Compound 5 was confirmed. The molecular formula was determined from the ion peak of 288.2085 observed at high resolution EI / MS (calcd. 288.2089 for C19H28O2) as C19H28O2. The NMR data of compound 5 was very similar to that of compound 1 and expected to be of the pimarane type. The oxygenated methine proton signal δH 5.48 (H-6) in the gCOSY showed a cross peak with the olefin methine proton signal at δH 5.70 (H-7) and methine proton signal δH 1.75 (H-5) δH 2.63 (H-4), which is a methine proton signal, and a cross peak. In the gHMBC, the methyl proton signal H-18 (δH 1.10) is the ketone carbon signal C-3 (δC 211.2) and H-6 (δH 5.48) is the olefin quaternary carbon signal C-8 (δC 144.8) (C-3) and hydroxyl group (C-6). The methylene carbon signals C-1 (δC 36.8), C-5, and methine carbon signals C-5 (δC 46.1) and methyl proton signal H-20 (δH 1.28) 9 (δC 50.5) and cross peak, and the methyl proton signal H-17 (δH 0.82) was the methylene carbon signal C-12 (δC 37.4), the olefin methine carbon signal C-15 (δC 149.7) (δC 21.8), C-18 (δC 11.5), and C-20 (δC 22.3)] with the carbon signal C-14 (δC 47.7). C-7 (隆 H 5.70) and C-9 (隆 H 2.05 and 1.89), which are methylene proton signals, are C-7 (隆 C 117.2), C- , C-17 and H-17 show cross peaks with C-12, C-14 and C-15, and the positions of double bonds are determined with C-7, C-8, C-15 and C-16 Respectively. The stereostructure of this compound is derived from the chemical shift and coupling pattern of all the chiral carbons except 6, and the moleilactone A (compound 1) and 3,6-dioxo-19-nor-9β-pimara-7,15-diene Respectively. In NOESY, δH1.10, which is H-18, was cross-peaked with δH 1.75, H-5, and δH 5.48, H-6, confirming that the C-6 hydroxyl group was β-bonded. Thus, compound 5 is a novel norditerpene compound, 3-oxo-6-beta-hydroxy-19-nor-9beta-pimarra-7,15-diene -9β-pimara-7,15-diene), and named as momilactone E.

Compound 2, on the other hand, confirmed the absorption of ketone (1706 cm ^-1 ) and double bond (1633 cm ^-1 ) in the IR spectrum. A 286 ion peak was observed in EI / MS and the molecular weight was determined to be 286. (1H, dd, J = 17.6, 10.8 Hz, H-15), 4.99 (1H, dd, J = 17.6, 1.2 Hz, H-16a) and 4.97 (1H, d, J = 10.8, 1.2 Hz, H-16b)], one olefin methine proton signal [δH5.35 (1H, br s, H-14)] and four methyl proton signals [δH1.13 (3H, s, H-19), 1.09 (3H, s, H-17), 0.99 (3H, s, H-18) and 0.89 Many methine and methylene proton signals were observed in the range of δH 2.60 to 0.80. Twenty carbon signals were observed in 13 C-NMR and compound 2 was expected to be a diterpenoid. (C-15), 129.7 (C-14), one olefin quaternary [δC 136.44 (C-8)], two olefin methane [δC 148.9 ], And one olefin methylene [δC 110.7 (C-16)] carbon signal was observed. In the high field region, two quaternary [δC 47.8 (C-4), 38.2 (C-13), and 37.8 (C-10)], two methane [δC 55.4 ), 6 methylene [delta C 37.6 (C-1), 35.8 (C-7), 34.8 (C-2), 34.4 (C-12), 23.5 , And four methyl [δC 26.4 (C-17), 26.1 (C-19), 22.2 (C-18), and 14.6 (C-20)] carbon signals were observed. From the above results, it was confirmed that Compound 2 is a diterpenoid containing one ketone and two double bonds. In the gHMBC, the ketone carbon signal, C-3 (δC 215.2), showed two methyl proton signals, H-18 (δH 0.99) and H-19 (δH 1.13). H-18 and H-19 showed a cross peak with the quaternary carbon signal C-4 (δC 47.8) and methine carbon signal C-5 (δC 55.4), and the methyl proton signal H-20 (δH 0.89) -5, the methine carbon signal C-9 (δC 49.5) and the quaternary carbon signal C-10 (δC 37.8) and the olefin methine proton signal H-14 (δH5.35) (C-17) (δ C 26.4), and the olefin methine proton signal, H-15 (δH5.84), showed a cross peak with C-17, ? C 26.4), C-18 (? C 22.2), C-19 (? C 26.1), and C-20 (? C 14.6). In addition, H-14 showed a cross peak with C-9, C-15 (δC 148.9) and C-17, H-15 showed C-14 (δC 129.7) C-8, C-14, and C-15 and C-16. CD spectra of oryzalexin A were compared to determine the exact structure of compound 2. Compound 2 exhibited a positive (203 nm) and negative (247 nm) Cotton effect, while oryzalexin A showed a negative (207 nm) and positive (255 nm) Cotton effects and compound 2 and oryzalexin A had the opposite structure . As a result of synthesis of the above results, Compound 2 was identified as sandaracopimaradien-3-one, a diterpenoid of the pimarane type.

On the other hand, Compound 4 confirmed the absorption of hydroxyl groups (3353 cm ^-1 ), conjugated ketone (1685 cm ^-1 ), and double bonds (1611 cm ^-1 ) in the IR spectrum. The ion peak of 302 was observed in EI / MS and the molecular weight was determined as 302. [ The NMR data of compound 4 was very similar to that of compound 2 and the binding sites of ketone (delta C 207.2) and hydroxyl groups (delta H 3.28, delta C80.5) were different. In the gHMBC, the methyl proton signals H-18 (δH 0.85) and H-19 (δH 0.96) showed cross-peaks with the hydroxyl carbon signal C-3 (δC 80.5) . In addition, the methylene proton signal H-6 (δH 2.54 and 2.34) showed a cross-peak with the ketone carbon signal C-7 (δC 207.2), confirming that ketone binds to C-7. The olefin methine proton signal H-14 (δH 6.71) showed a cross peak with the methine carbon signal C-9, the methylene carbon signal C-12 and the olefin methine carbon signal C-15, and the methyl proton signal H-17 showed cross peaks with C-14 and C-15. H-16 (δH 4.98 and 4.96) olefin methylene carbon signals showed cross peaks with C-13 (δC 38.7) quaternary carbon signal Of the double bonds were located at C-8 and C-14, and at C-15 and C-16. Therefore, Compound 4 was identified as oryzalexin A.

&Lt; Compound 1 >

<Compound 2>

<Compound 3>

<Compound 4>

<Compound 5>

The spectroscopic characteristics of the compounds 1 to 5

Marmalade lactone ( momilactone  A) (Compound 1)

Colorless crystals (CHCl3); [[alpha]] D &lt; 20 &gt; -275 [deg.] (c 1.0, CHCl3); EI / MS m / z 314 [M] &lt; + &gt;; ^{IR (KBr, ν, cm -1} ) 1770, 1700, 1665, 1640; 1H NMR (400 MHz, CDCl3, δH) 5.79 (1H, dd, J = 17.6, 10.8 Hz, H-15), 5.64 (1H, d, J = 5.8 Hz, H-7), 4.92 (1H, dd, J = 17.6,1.2 Hz, H-16a), 4.87 (2H, m, H-2), 2.29 (1H, d, J = 5.2 Hz, H-5), 2.15 (1H, br d, (1H, m, H-9), 1.69 (1H, m, H-12) (1H, m, H-11), 1.56 (1H, m, H-1b), 1.53 -11b), 1.25 (3H, s, H-20), 0.83 (3H, s, H-17). C-15, C-14, C-14, C-13, C-17, C- ), 73.0 (C-6), 53.4 (C-4), 50.0 (C-9), 47.4 (C-14), 46.2 , 34.7 (C-2), 32.3 (C-10), 31.0 (C-1), 23.8 (C-11), 21.8 (C-17), 21.6 (C-20), 21.3 (C-18).

Sandarakopi Mara Dien 3-one ( sandaracopimaradien -3- one ) (Compound 2)

White powder (CHCl3); ^{[α] 32 D + 2.4 °} (c 0.2, MeOH); EI / MS m / z 286 [M] +; IR (KBr,?, Cm ^-1 ) 1706, 1633; 1 H NMR (400 MHz, C5D5N, H-16b), 4.97 (1H, d, J = 10.8, 1.2 Hz, H-16b) ), 2.60 (1H, ddd, J = 15.2,14.0,6.0 Hz, H-2a), 2.31 (1H, ddd, J = 15.2, 4.4, 4.4 Hz, H- 19), 1.09 (3H, s, H-17), 0.99 (3H, s, H-18), 0.89 (3H, s, H-20). (C-15), 136.44 (C-8), 129.7 (C-14), 110.7 (C-16), 55.4 (C-5) ), 49.8 (C-9), 47.8 (C-4), 38.2 (C-13), 37.8 , 34.4 (C-12), 26.4 (C-17), 26.1 (C-19), 23.5 (C-6), 22.2 (C-18), 19.2 (C-11), 14.6 (C-20).

Marmalade lactone  D ( momilactone  D) (Compound 3)

White powder (CHCl3-MeOH); _{^{[α] 27 D -57.1 ° (}} c 0.46, pyridine); HREI / MS m / z 330.1833 [M] + (calcd. For C20H26O4, 330.1831); (KB, v, cm ^-1 ) 3374, 2918, 2345, 1767, 1699, 1647, ), 5.81 (1H, d, J = 5.2 Hz, H-7), 5.04 (1H, dd, J = 17.2,1.2 Hz, H- D, J = 10.2 Hz, H-16b), 3.18 (1H, d, J = 11.2 Hz, H-14a), 2.73 (1H, ddd, J = 13.2, 1H), 2.63 (1H, ddd, J = (1H, m, H-1b), 1.83 (1H, m, H-11a), 1.78 H-12b), 1.37 (1H, m, H-12b), 1.72 (1H, br dd, J = 11.2 Hz, H- , 1.32 (3H, s, H-20), 0.94 (3H, s, H-17). C-15), 149.6 (C-8), 116.9 (C-7), 110.0 (C-16) ), 74.4 (C-9), 72.5 (C-6), 54.7 (C-4), 48.4 , 35.3 (C-2), 32.8 (C-12), 30.0 (C-11), 27.8 (C-1), 21.9 (C-17), 21.2 (C-18), 17.3 (C-20).

Dorydalin  A ( oryzalexin  A) (Compound 4)

White powder (CHCl _3); [?] ²⁵ _D +20 (c 0.13, MeOH); EI / MS m / z 302 [M] &lt; + &gt;,164; ^{IR (KBr, ν, cm -1} ) 3353, 1685, 1611; 1H NMR (400 MHz, CDCl3, δH) 6.71 (1H, d, J = 2.4 Hz, H-14), 5.78 (1H, dd, J = (1H, dd, J = 10.8, 1.2 Hz, H-16b), 3.28 (1H, dd, J = 11.6, 4.0 Hz, H-3), 2.54 (1H, dd, J = 18.8, 5.2 Hz, H- M, H-11a), 1.68 (1H, m, H-9), 1.80 (1H, ddd, J = 13.2, 3.6, 3.2 Hz, H- M, H-2b), 1.59 (1H, m, H-12a), 1.49 H-11b), 1.28 (1H, m, H-1b), 1.08 (3H, s, H-17), 0.96 ), 0.82 (3H, s, H-20). (C-15), 144.5 (C-14), 134.7 (C-8), 111.8 (C-16), 80.5 ), 50.6 (C-9), 49.5 (C-5), 38.9 (C-4), 38.7 12), 27.4 (C-19), 27.3 (C-2), 25.8 (C-17), 19.1 (C-11), 14.6 (C-18), 13.8 (C-20).

Marmalade lactone  E ( momilactone  E) (Compound 5)

Yellow amorphous powder (CHCl3); _{^{[α] 27 D -101 ° (}} c 0.18, MeOH); HREI / MS m / z 288.2085 [M] + (calcd for C19H28O2, 288.2089); ^{IR (KBr, ν, cm -1} ) 3620, 1705, 1614; 1H NMR (400 MHz, C5D5N, δH) 5.84 (1H, dd, J = 17.6, 10.8 Hz, H-15), 5.70 (1H, d, J = 5.2 Hz, H-7), 5.48 (1H, dd, J = 5.2, 5.2 Hz, H-6), 4.99 (1H, dd, J = dd, J = 10.8,1.2 Hz, H-16b), 2.63 (1H, dq, J = 11.6, 6.8 Hz, H-4), 2.58 (1H, d, J = 12.0 Hz, H), 2.38 (1H, ddd, J = 15.2,3.6,3.2 Hz, H- (1H, d, J = 11.6, 5.2 Hz, H-5), 1.67 J = 6.8 Hz, H-18), 0.82 (3H, s, H-17). (C-15), 144.8 (C-8), 117.2 (C-7), 110.3 (C-16), 67.7 (C-6) ), 37.9 (C-2), 37.4 (C-12), 50.9 (C-9) , 36.8 (C-1), 34.0 (C-10), 24.3 (C-11), 22.3 (C-20), 21.8 (C-17), 11.5 (C-18).

<Experimental Example 1> Nitric oxide production and cytotoxicity evaluation of rice root extract / fraction

The effects of methanol extracts and fractions of rice seeds of Examples 1 to 5 on the production of nitric oxide in LPS-stimulated RAW264.7 cells were analyzed. For this purpose, various concentrations of samples were prepared and tested.

As a result, the most potent inhibitory effect on nitric oxide formation was found in the rice seed methanol extract (Example 1) and the ethyl acetate fraction (Example 2) (FIG. 1). In addition, Example 1 and Example 2 showed cell viability of 80% or more even at a concentration strongly inhibiting nitric oxide production (FIG. 2). As a result, it was found that the inhibitory activity against nitric oxide formation was not the result of cell death.

<Experimental Example 2> Evaluation of nitric oxide production and cytotoxicity of rice root-derived compounds

Dipterpen compounds isolated from rice roots were treated at various concentrations and their effects on nitric oxide production were analyzed.

As a result, the diterpene compounds inhibited nitric oxide production in a concentration-dependent manner (Fig. 3), and IC50 values were as shown in Table 1 below. In addition, the effects of the diterpene compounds on the survival and death of the cells were analyzed. As a result, Compound 2 showed weak cytotoxicity (LD50, 83.2 ± 3.0 μM). On the other hand, it was confirmed that Compound 1 exhibited strong cytotoxicity (LD50, 9.6 + - 0.6 [mu] M) (Table 2, Fig.

^a IC ₅₀ means the inhibition concentration, which means a concentration indicating a 50% inhibition rate.

^b LD ₅₀ means the lethal dose, which represents the 50% survival rate.

<Experimental Example 3> Inhibition of iNOS expression in rice root-derived compounds

Compounds 1 to 5 were treated with RAW264.7 cells with 1 μg / mL of LPS at various concentrations or treated with LPS alone without compound and incubated for 18 hours. The results were analyzed by reverse transcriptase - polymerase chain reaction and Western blot analysis.

As a result, it was confirmed that the compounds 1 and 4 strongly inhibited the expression of mRNA and protein of iNOS in both the reverse transcriptase-polymerase chain reaction analysis result and the Western blot analysis result (FIGS. 5A and 5B, respectively). From the above results, it was confirmed that the diaperpen compounds of the compounds 1 to 5 isolated from the rice seeds weakened the inflammatory reaction.

Claims (11)

A pharmaceutical composition for the prophylaxis or treatment of inflammatory diseases, which comprises a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof:

&Lt; Formula 1 >

Wherein R 1 is H or OR 2, and R ₂ is selected from the group consisting of H, CH ₃ , CH ₂ CH ₃ and CH ₂ CH ₂ CH ₃ .

(2)

(3)

&Lt; Formula 4 >

In this case, R3 is H or OR4, wherein R4 is is one which is selected from the group consisting of _{H, CH 3, CH 2 CH} 3 and CH ₂ CH ₂ CH _3.
The method according to claim 1,
Wherein said pharmaceutical composition inhibits the expression of nitric oxide (NO).
The method according to claim 1,
Wherein the inflammatory disease is selected from the group consisting of edema, dermatitis, allergy, atopy, asthma, conjunctivitis, periodontitis, rhinitis, otitis, sore throat, tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn's disease, colitis, hemorrhoids, Wherein the composition is any one selected from the group consisting of fibromyalgia, psoriatic arthritis, osteoarthritis, rheumatoid arthritis, shoulder joint inflammation, tendonitis, nephritis, myositis, hepatitis, cystitis, nephritis and multiple sclerosis.
A food composition for preventing or ameliorating inflammation comprising a compound represented by the following formula (1) or an acceptable salt thereof:

&Lt; Formula 1 >

Wherein R 1 is H or OR 2, and R ₂ is selected from the group consisting of H, CH ₃ , CH ₂ CH ₃ and CH ₂ CH ₂ CH ₃ .

(2)

(3)

&Lt; Formula 4 >

In this case, R3 is H or OR4, wherein R4 is is one which is selected from the group consisting of _{H, CH 3, CH 2 CH} 3 and CH ₂ CH ₂ CH _3.
A cosmetic composition for preventing or ameliorating inflammation comprising a compound represented by the following formula (1) or an acceptable salt thereof:

&Lt; Formula 1 >

Wherein R 1 is H or OR 2, and R ₂ is selected from the group consisting of H, CH ₃ , CH ₂ CH ₃ and CH ₂ CH ₂ CH ₃ .

(2)

(3)

&Lt; Formula 4 >

In this case, R3 is H or OR4, wherein R4 is is one which is selected from the group consisting of _{H, CH 3, CH 2 CH} 3 and CH ₂ CH ₂ CH _3.
A compound represented by the following formula (1) or an acceptable salt thereof:

&Lt; Formula 1 >

Wherein R 1 is OR 2 and R ₂ is selected from the group consisting of H, CH ₃ , CH ₂ CH ₃ and CH ₂ CH ₂ CH ₃ .
The method according to claim 6,
Wherein said compound has the ability to prevent, improve or treat inflammation, or an acceptable salt thereof.
A method for producing the compound of claim 6, comprising the step of extracting the rice with an organic solvent.
A compound represented by the following formula (4) or an acceptable salt thereof:

&Lt; Formula 4 >

In this case, R3 is H or OR4, wherein R4 is is one which is selected from the group consisting of _{H, CH 3, CH 2 CH} 3 and CH ₂ CH ₂ CH _3.
10. The method of claim 9,
Wherein said compound has the ability to prevent, improve or treat inflammation, or an acceptable salt thereof.
9. A method for producing a compound according to claim 9, comprising the step of extracting the rice with an organic solvent.

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