KR20120089893A - Pharmaceutical compositions for prevention and treatment of inflammatory diseases containing the aerial parts of oryza sativa l. extracts, fractions, the isolated flavonolignans compounds therefrom, or the pharmaceutically acceptable salts as an active ingredient - Google Patents

Abstract

PURPOSE: A pharmaceutical composition containing extract and fraction of an aerial part of Oryza sativa L. and flavonolignan compounds isolated from the same is provided to suppress NO generation and to reduce side effects. CONSTITUTION: A pharmaceutical composition for preventing and treating inflammatory diseases contains extract of an aerial part of Oryza sativa L. as an active ingredient. The extract is prepared using 80% methanol solution. The pharmaceutical composition contains a fraction of an aerial part of Oryza sativa L. as an active ingredient. The fraction is prepared by extracting the extract using n-hexane and H_2O, extracting a H_2O layer using ethyl acetate, and extracting H_2O by n-butanol. The pharmaceutical composition contains flavonolignan compounds or pharmaceutically acceptable salt thereof as an active ingredient.

Description

Pharmaceutical composition for the prevention and treatment of inflammatory diseases containing rice ground extract, rice ground fraction, flavonolignan compound isolated therefrom or a pharmaceutically acceptable salt thereof as an active ingredient {PHARMACEUTICAL COMPOSITIONS FOR PREVENTION AND TREATMENT OF INFLAMMATORY DISEASES CONTAINING THE AERIAL PARTS OF Oryza sativa L. EXTRACTS, FRACTIONS, THE ISOLATED FLAVONOLIGNANS COMPOUNDS THEREFROM, OR THE PHARMACEUTICALLY ACCEPTABLE SALTS AS AN ACTIVE INGREDIENT}

The present invention relates to a pharmaceutical composition for the prevention and treatment of inflammatory diseases containing rice ground extract, rice ground fraction, flavonolignan compound isolated therefrom or a pharmaceutically acceptable salt thereof as an active ingredient.

Inflammation causes a variety of diseases, including rhinitis, bronchitis, hepatitis, and arthritis, so close to our health that no one has ever had one or two inflammatory diseases in their lifetime. Has a connection. Especially in the case of degenerative diseases in which the problem is serious due to the aging of the population due to prolonged life expectancy, interest in the mechanism and role of the inflammatory response has sharply increased as research reports show that the pathophysiology is closely related to the inflammatory and immune response. have.

NO (Nitric Oxide), one of the inflammatory substances, is produced in endothelial cells or macrophages in a normal state and exhibits various physiological activities in addition to cell killing and bactericidal action. In particular, homeostasis of blood pressure in the relaxation of vascular endothelial cells It is known to play a role in maintaining). Stimulation, such as LPS, proinflammatory factors and irradiation, in particular, induces the expression of an inducible nitric oxide synthase (iNOS), which produces a large amount of NO continuously for 4-6 hours. Excessive amounts of NO may lead to such diseases.

Therefore, the development of an iNOS activity inhibitor is likely to be developed as a therapeutic agent for the above-mentioned diseases, and in this respect, compounds that inhibit NO production by iNOS can be used as therapeutic agents for various inflammatory diseases in the human body. Prostaglandins, such as PGE2, PGF2a and PGI2, are other hormone-derived hormones derived from arachidonic acid and are involved in various physiological activities and are regulated by cyclooxygenase (COX). Expression is regulated. Nonsteroidal anti-inflammatory drugs, such as aspirin indomethacin, which inhibit the synthesis of prostaglandins by inhibiting COX activity, are known to have excellent effects on arthritis.

Finally, TNF-α is produced in macrophages and fibroblasts activated by BCG, LPS and proinflammatory factors, leading to cytotoxic effects and production of prostaglandins and cytokines such as IL-1 and IL-6. . TNF-α, a tumor necrosis factor, has been reported to be related to autoimmune diseases such as rheumatoid arthritis and organ transplant rejection, allergic inflammatory diseases such as asthma and atopic dermatitis, and acute immune diseases such as pneumonia and acute liver disease. As a result, research into compounds that inhibit the synthesis of TNF-α is being actively conducted.

Meanwhile, Oryza sativa L.) is a herbaceous herbaceous plant belonging to Gramineae and is widely distributed throughout Asia. Its height is 50-100 cm and grows in the form of aeration by branching near the roots. When flowers bloom, they stand upright, but when they ripen, they fall down, and many of them run, and a few live on branches. Flowers bloom in July-August and fruit by self pollination.

Research on rice has been mainly focused on improving productivity by breeding and increasing resistance to pest diseases in agricultural cultivation. Several types of steroids, terpenoids and alkaloids have been reported from rice, including ergosterol peroxide and 7-oxo-stigmasterol. Has been reported to have phytotoxic effects. Extract from the bark of rice chaff is typically a 1-tetrahydro tree Acorn butanol (1-tetratriacontanol), β - sitosterol (β -sitosterol), Mo mirak ton A (momilactone A), parent mirak tone B (momilactone B), Tricin has been reported. Momiractone A has been reported to have herbicide activity and Momiractone B has been reported to have anticancer activity. However, little research has been conducted on the above-ground part of rice, and no NO production inhibitory activity of flavonolignan compounds extracted from the above-ground part of rice has been reported.

Therefore, the present inventors were studying rice ground extract, rice ground fraction, and flavonolignan compounds isolated therefrom, and the rice ground extract, rice ground fraction, and flavonolignan compounds isolated therefrom had NO production inhibitory activity. The present invention was completed by revealing the use for preventing and treating these inflammatory diseases.

It is an object of the present invention to provide a pharmaceutical composition for the prevention and treatment of inflammatory diseases, containing as an active ingredient a rice ground extract, rice ground fraction, flavonolignan compound or pharmaceutically acceptable salts thereof.

Another object of the present invention is to prevent and treat inflammatory diseases that may reduce side effects by containing natural ingredients such as rice ground extract, rice ground fraction, flavonolignan compounds isolated therefrom or pharmaceutically acceptable salts thereof. It is to provide a pharmaceutical composition for.

In order to achieve the above object, the present invention provides a pharmaceutical composition for the prevention and treatment of inflammatory diseases containing rice ground extract, rice ground fraction, flavonolignan compound isolated therefrom or a pharmaceutically acceptable salt thereof as an active ingredient. To provide a composition.

In one embodiment of the present invention, the above-ground rice extract may be obtained by extracting the above-ground rice body living body with an aqueous alcohol solution.

In one embodiment of the present invention, the above-ground rice fraction fraction is extracted by extracting the above-ground rice extract with n -hexane and H ₂ O, and extracted the H ₂ O layer with ethyl acetate, the H ₂ O layer to n -butanol Can be obtained by partition extraction.

In one embodiment of the present invention, the flavonolignan compound is a step of extracting the rice ground body living body with an aqueous alcohol solution to obtain a rice ground extract; Of the rice shoot extract rice shoot extract n - hexane and then extract partitioned between H ₂ O and extracted distribute the H ₂ O layer with ethyl acetate, the H ₂ O layer n - extracts partitioned between butanol to obtain a rice plant aboveground fraction And rice pad fractions can be obtained by performing a step of separating and purifying the compound by silica gel column chromatography.

The inflammatory disease may be rhinitis, bronchitis, arthritis, sepsis, periodontitis, gastric ulcer, pancreatitis, inflammatory skin disease, inflammatory bowel disease, and the like.

The pharmaceutical composition according to the present invention has an activity of inhibiting the production of nitric oxide (NO), which is one of the inflammation-inducing substances.

The pharmaceutical composition for the prevention and treatment of inflammatory diseases containing rice ground extract, rice ground fraction, flavonolignan compound or pharmaceutically acceptable salt thereof as an active ingredient according to the present invention is one of inflammatory substances By having a production inhibitory activity against phosphorous NO (Nitric Oxide) it is possible to significantly reduce the side effects compared to the conventional inflammatory disease treatment.

Figure 1 is a graph showing the cell survival rate measured by cytotoxicity test for cell cultures treated with Salcorin B in Experimental Example 1 of the present invention.
Figure 2 is a graph showing the cell viability measured by the cytotoxicity test for cell cultures treated with Salcorin C in Experimental Example 1 of the present invention.
Figure 3 is a graph showing the cell viability measured by the cytotoxicity test for the cell culture solution treated with Salcorinoside A in Experimental Example 1 of the present invention.
Figure 4 is a graph showing the cell viability measured by the cytotoxicity test for the cell culture medium treated with Salcorinoside B in Experimental Example 1 of the present invention.
FIG. 5 is a graph showing cell viability measured by cytotoxicity test on a cell culture treated with saccharinoside C in Experimental Example 1 of the present invention. FIG.
Figure 6 is a graph showing the results of measuring the nitric oxide inhibitory activity of the cell culture medium treated with Salcorin B in Experimental Example 1 of the present invention.
Figure 7 is a graph showing the results of measuring the nitrogen monoxide inhibitory activity of the cell culture medium treated with Salcorin C in Experimental Example 1 of the present invention.
Figure 8 is a graph showing the results of measuring the nitrogen monoxide inhibitory activity for the cell culture solution treated with Salcorinoside A in Experimental Example 1 of the present invention.
9 is a graph showing the results of measuring the nitric oxide inhibitory activity of the cell culture medium treated with Salicynoside B in Experimental Example 1 of the present invention.
Figure 10 is a graph showing the results of measuring the nitrogen monoxide inhibitory activity for the cell culture solution treated with Salcorinoside C in Experimental Example 1 of the present invention.

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for the prophylaxis and treatment of inflammatory diseases containing the extract of the ground rice as an active ingredient.

The above-ground rice extract may be obtained by extracting the above-ground rice biomass with an aqueous alcohol solution, such as 80% methanol aqueous solution, and the above-ground rice biomass may be used without limitation, such as those grown or commercially available. The method of preparing the above-ground rice extract may use a conventional extraction method such as ultrasonic extraction, filtration, reflux extraction method.

In one embodiment of the present invention, the above-ground rice extract may be obtained by putting the above-ground rice extract into 80% aqueous methanol solution to obtain an extract, followed by filtration and concentration under reduced pressure.

In addition, the present invention provides a pharmaceutical composition for the prevention and treatment of inflammatory diseases containing the above-ground fraction of rice as an active ingredient.

The above-ground rice fraction was extracted by extracting the above-ground rice extract obtained as described above with n -hexane and H ₂ O, extracting the H ₂ O layer with ethyl acetate (EtOA), and then extracting the H ₂ O layer with n -butanol. By partition extraction, n -hexane fraction, ethyl acetate fraction, n -butanol fraction and H ₂ O fraction can be obtained.

The present invention also provides a pharmaceutical composition for the prevention and treatment of inflammatory diseases containing the flavonolignan compound or a pharmaceutically acceptable salt thereof obtained by fractionation from the above-ground rice fraction as an active ingredient.

The flavonolignan compound is salcolin B represented by the following formula (1), salcolin C (salcolin C) represented by the formula (2), salcolinoside A (salcolinoside A) represented by the formula (3), It may be represented by salcolinoside B or salcolinoside C represented by the formula (5).

[Formula 1]

[Formula 2]

(3)

[Formula 4]

[Chemical Formula 5]

In the present invention, the flavonolignan compound represented by Formula 1 to Formula 5 may be obtained by separating and purifying the ethyl acetate fraction obtained as described above by silica gel column chromatography.

When silica gel column chromatography is performed to separate the flavonolignan compound represented by Formula 1 to Formula 5 from the ethyl acetate fraction, n -hexane, ethyl acetate, methanol mixed solvent and CHCl ₃ . Can be used. The silica gel column chromatography may be performed once to several times until a single compound is purified, and may be concentrated and recrystallized as necessary.

A pharmaceutical composition containing a rice ground extract, a rice ground fraction, a flavonolignan compound isolated therefrom or a pharmaceutically acceptable salt thereof as an active ingredient prepared as described above, inhibits NO production from cells. Having activity can be used for the prevention and treatment of anti-inflammatory diseases.

In the present invention, the flavonolignan compound represented by Formula 1 to Formula 5 may be used in the form of a pharmaceutically acceptable salt, and the salt may be an acid addition salt formed by a pharmaceutically acceptable free acid. useful. Acid addition salts include those derived from inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid or phosphorous acid, and aliphatic mono- and dicarboxylates, phenyl-substituted alkanoates, hydroxyalkanoates, Dioleate, aromatic acid, aliphatic and aromatic sulfonic acids. Such pharmaceutically innocuous salts include, but are not limited to, sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate chloride, bromide, Butyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, succinate, maleic anhydride, maleic anhydride, , Sebacate, fumarate, maleate, butyne-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, Methoxybenzoate, phthalate, terephthalate, benzene sulfonate, toluene sulfonate, chlorobenzene sulfide Propyl sulphonate, naphthalene-1-yne, xylenesulfonate, phenylsulfate, phenylbutyrate, citrate, lactate,? -Hydroxybutyrate, glycolate, maleate, Sulfonate, naphthalene-2-sulfonate or mandelate.

The acid addition salt according to the present invention is dissolved in a conventional method, for example, the flavonolignan compound represented by the above formulas (1) to (5) in an excess of an aqueous acid solution, and the salt is a water miscible organic solvent such as methanol. It can be prepared by precipitation with ethanol, acetone or acetonitrile.

The same amount of flavonolignan compound represented by the above formulas (1) to (5) may be prepared by heating an acid or an alcohol and then evaporating the mixture to dry it to prepare a salt or a precipitated salt by suction filtration.

In addition, bases can be used to make pharmaceutically acceptable metal salts. Alkali metal or alkaline earth metal salts are obtained, for example, by dissolving a compound in an excess of alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the insoluble compound salt, and evaporating and drying the filtrate. At this time, it is pharmaceutically suitable to prepare sodium, potassium or calcium salt as the metal salt. Corresponding salts may also be obtained by reacting alkali or alkaline earth metal salts with a suitable negative salt (eg, silver nitrate).

The composition may be used as a pharmaceutical composition and may be various oral dosage forms. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid form preparations for oral administration include tablets, pills, powders, granules, capsules and the like, which form at least one excipient such as starch, calcium carbonate, sucrose or lactose in one or more compounds. (lactose), gelatin, etc. are mixed and prepared. In addition to simple excipients, lubricants such as magnesium stearate, talc and the like are also used.

The pharmaceutical composition comprising the flavonolignan compound represented by Formula 1 to Formula 5 of the present invention as an active ingredient is not mixed well when water, liquid paraffin, or the like, which is a diluent, is used in suspensions, solvents, emulsions, syrups, and the like. Therefore, it is not suitable as a liquid preparation for oral administration. However, the pharmaceutical composition containing the above-ground rice extract or the above-ground rice fraction as an active ingredient may be prepared as a liquid preparation such as suspending agents, liquid solutions, emulsions, and syrups.

Hereinafter, the present invention will be described in detail through Examples and Experimental Examples. However, the following examples are illustrative of the present invention, and the present invention is not limited by the following examples.

Example  1: Preparation of Rice Ground Extract

Cheongcheongpum collected from Bubal-eup, Icheon-si, Gyeonggi-do, was used as a rice ground biomaterial, and 26.7 kg of the above-ground rice biomass was extracted in an aqueous 80% MeOH solution (18 L) at room temperature for 24 hours. The obtained extract was filtered and extracted twice in the same manner. The obtained extracts were collected by filtration and concentrated under reduced pressure to obtain a MeOH extract.

Example  2: rice ground Fraction  Produce

The MeOH extract was partitioned and extracted with n -hexane (3 L × 3) and H ₂ O (3 L), and the H ₂ O layer was extracted with ethyl acetate (EtOAc, 3 L × 3). Again H ₂ O layer was obtained by partitioning with n -butanol ( n- BuOH, 3 L × 3), each layer was concentrated under reduced pressure, n -hexane fraction (135 g, OSH), ethyl acetate fraction (47 g, OSE), n -butanol fraction (90 g, OSB) and H ₂ O fraction.

Example  3: rice ground From fractions Flavono League  Isolation of the compound

Silica gel column chromatography (cc) (φ 12 × 20 cm, n -hexane-EtOAc-MeOH = 25: 3: 1 → 23: 3: 1 → CHCl ₃ -MeOH = 20: from the ethyl acetate fraction obtained in Example 2): 1 → 18: 1 → 16: 1 → 13: 1 → 7: 1 → 5: 1 → 3: 1 → 1: 1), and 70 ml were separated. Each aliquot was identified by SiO ₂ TLC, similar portions were collected together and concentrated to give 17 fractions (OSE-1 to OSE-17). Among them, OSE-5 fraction (3.5 g) was subjected to silica gel column chromatography (Kiesel gel 60, Merck Darmstadt, Germany) (silica gel cc) (φ 5.5 × 14 cm, CHCl ₃ -MeOH = 50: 1). Fractions (OSE-1 to OSE-15) were obtained, of which OSE-5-7-9 fractions (573 mg) were subjected to octadecyl silica gel column chromatography (ODS cc) (φ 3 cm × 6 cm, MeOH). -H ₂ O = 1: 1) to obtain 14 fractions (OSE-5-7 ~ 9-1 ~ OSE-5-7 ~ 9-14). Among them, OSE-5-7 to 9-5 to 8 fractions (111 mg) were subjected to ODS cc (φ 3 cm × 5 cm, MeOH-H ₂ O = 3: 2) to 9 fractions (OSE-5- 7 to 9-8 to 10-1 to OSE-5-7 to 9-5 to 8-9), and among these, salcolin B represented by Formula 1 [OSE-5-7? 9] _-8-10-10 , 24 mg, TLC (RP-18 F _254S ) R _f 0.4, MeOH-H ₂ O = 3: 1].

Thereafter, OSE-6 fraction (4.2 g) was subjected to silica gel column chromatography (cc) (φ 5.5 cm × 15 cm, CHCl ₃ -MeOH = 15: 1) to obtain nine fractions (OSE6-1 to OSE-6). -9) was obtained. OSE-6-2 fraction (210 mg) was subjected to ODS cc (φ 3.5 × 6 cm, MeOH-H ₂ O = 1: 1) to 13 fractions (OSE-6-2-1 to OSE-6-2). -13), of which OSE-6-2-10-12 fractions (103 mg) were subjected to silica gel column chromatography (cc) (φ 3 cm × 13 cm, CHCl ₃ -MeOH-H ₂ O = 26: 3: 1) and divided into six fractions (OSE-6-2-10-12-1 ~ OSE-6-2-10-12-12-6), of which OSE-6-2-10-12-12- One to three fractions (57 mg) were subjected to ODS cc (φ 3 × 5 cm, MeOH-H ₂ O = 2: 1) to seven fractions (OSE-6-2-10 to 12-1 to 3-1 OSE-6-2-10 to 12-1 to 3-7), and among them, salcolin C represented by Chemical Formula 2 [OSE-6-2-10 to 12-1 to 3- 4 + 5, 24 mg, TLC (RP-18 F _254S ) R _f 0.5, MeOH-H ₂ 0 = 4: 1].

Then, 14 fractions (OSE-12-1 to OSE-12-14) were subjected to ODS cc (φ 5.5 cm × 7 cm, MeOH-H ₂ O = 2: 3) with respect to the OSE-12 fraction (1.96 g). ) OSE-12-9 fraction (80 mg) was subjected to ODS cc (φ 3.5 × 6 cm, MeOH-H ₂ O = 1: 1) to 12 fractions (OSE-12-9-1 to OSE-12-9). -12), wherein salcolinoside A represented by the formula (3) [OSE-12-9-10, 20 mg, TLC (RP-18 F _254S ) R _f 0.5, MeOH-H ₂ 0 = 1: 1].

Thereafter, OSE-14 fraction (1.4 g) was subjected to ODSc.c. (φ 4 × 6 cm, MeOH-H ₂ 0 = 1.5: 1) to 18 fractions (OSE-14-1 to OSE-14-). 17), and OSE-14-11 fraction (152 mg) was subjected to ODS cc (φ 3.5 × 6 cm, MeOH-H ₂ O = 1: 1) to eleven fractions (OSE-14-11- 1 to OSE-14-11-11), and among these, salcolinoside B (salcolinoside B) represented by Chemical Formula 4 [OSE-14-11-6, 34 mg, TLC (RP-18 F _254S ) R _f 0.5, MeOH-H ₂ 0 = 1: 1].

Thereafter, OSE-15 fraction (1.82 g) was subjected to ODS cc (φ 4.5 cm × 7 cm, MeOH-H ₂ O = 1: 4) to 20 fractions (OSE-15-1 to OSE-15-20). ) OSE-15-15 fraction (230 mg) was subjected to ODS cc (φ 3.5 × 6 cm, MeOH-H ₂ O = 1: 2 → 3: 2) to 12 fractions (OSE-15-15-1 ~ OSE -15-15-12), of which salcolinoside C (salcolinoside C) represented by Formula 5 [OSE-15-15-12, 19 mg, TLC (RP-18 F _254S ) R _f 0.5, MeOH-H ₂ O = 3: 2].

Test Example  One: Flavono League  Structural analysis of compounds

For the flavonolignan compound represented by Formula 1 to Formula 5 obtained in Example 3, the IR spectrum was measured using a Perkin model 599B (Buckinghamshire, England), the NMR is 400 MHz FT-NMR spectrometer ( a AS 400, FT-NMR spectrometer (Varian Inova AS 400, Palo Alto, Calif.), fast atom bombardment mass spectrum was measured using JMSAX 700 (JEOL), UV UV lamps were measured using spectroline (Spectronics Corporation, Westbury, NY), non-luminescence was measured using polarimeter P-1020 (JASCO, Tokyo, Japan), melting point was Fisher-John's Melting Point Apparatus (Fisher Scientific) , Miami, FL, USA) was used to measure the structure and the results are as follows.

(One) Salmon  B ( salcolin  B)

[Formula 1]

1) yellow powder

2) [α] ²⁴ _D + 15 ° ( c = 0.05, MeOH); IR (MeOH, cm ⁻¹ ): 3457, 2939, 2640, 2558, 1653, 1591, 1498, 1354; Negative FAB / MS m / z : 525 [M ⁻ H] ⁻ , 329, 297, 151; ¹ H-NMR (400 MHz, CD ₃ OD); 7.07 (H-2 ', 6', s), 6.56 (H-3), 6.36 (H-8), 6.11 (H-6), 6.99 (H-2 ", d, J = 1.6 Hz), 6.74 (H-5 ", d, J = 8.0 Hz), 6.82 (H-6", dd, J = 8.0, 1.6 Hz), 4.94 (H-7 ", d, J = 5.2 Hz), 4.41 (H- 8 ", dd, J = 5.2, 3.6 Hz), 3.93 (H-9" a, dd, J = 12.0, 5.6 Hz), 3.65 (H-9 "b, dd, J = 12.0, 3.0 Hz), 3.86 (H-3 ', 5', s), 3.83 (H-3 ", s); ¹³ C-NMR (100 MHz, CD ₃ OD); 183.4 (C-4), 166.0 (C-7), 164.7 (C-2), 162.9 (C-5), 159.1 (C-9), 154.5 (C-3 ', 5'), 148.4 (C- 3 "), 146.7 (C-4"), 140.3 (C-4 '), 133.6 (C-1 "), 127.4 (C-1'), 105.3 (C-10), 120.6 (C-6") , 115.5 (C-5 "), 111.3 (C-2"), 105.5 (C-3), 104.7 (C-2 ', 6'), 100.1 (C-6), 95.1 (C-8), 87.47 (C-8 "), 74.16 (C-7"), 61.8 (C-9 "), 56.8 (C-3 ', C-5'), 56.3 (C-3").

3) Analysis result

Salmonine B isolated in Example 3 was observed to develop on TLC, and the result was UV absorption, and yellow color appeared when sprayed with 10% sulfuric acid and heated. The peak of m / z 525 [MH] ⁻ was observed in FAB / MS, and the molecular weight was determined to be 526 [M + 1] ⁺ . In the IR spectrum, the hydroxyl group (3484 cm ⁻¹ ) and the double bond (1640 cm ⁻¹ ) were observed. Confirmed its presence. Five singlet methyl proton signals observed in the low-field region of the ¹ H-NMR (400 MHz, CD ₃ OD, δ _H ) spectrum [δ _H 7.07 (H-2 ', 6', s ), 6.56 (H-3), 6.36 (H-8), 6.11 (H-6)]. Also, two doublet methyl proton signals (δ _H 6.99 (H-2 ", d, J = 1.6 Hz), 6.74 (H-5", d, J = 8.0 Hz)] and one The presence of 1,2,4-3 substituted benzene rings was confirmed from the doublet olefin methine proton signal [δ _H 6.82 (H-6 ”, dd, J = 8.0, 1.6 Hz)]. Oxygenated methine proton signal (δ _H 4.94 (H-7 ", d, J = 5.2 Hz), 4.41 (H-8", dd, J = 5.2, 3.6 Hz)] and one methine oxide Proton signal [δ _H 3.93 (H-9 ”a, dd, J = 12.0, 5.6 Hz), 3.65 (H-9” b, dd, J = 12.0, 3.0 Hz)], two methyl oxide proton signals [δ 3.86 (H-3 ', H-5'), 3.83 (H-3 ")]. ^{In the 13} C-NMR (100 MHz, CD ₃ OD, δc) spectrum, 24 signals other than 3 methoxy were observed, and DEPT experiments determined the multiplicity of each carbon. Conjugated ketone (δc 183.4) and nine olefin oxide quaternary carbon signals in the low-field region [δc 166.0 (C-7), 164.7 (C-2), 162.9 (C-5), 159.1 (C-9), 154.5 (C-3 ', C-5'), 148.4 (C-3 "), 146.7 (C-4"), 140.3 (C-4 ')] were observed and three olefin quaternary carbon signals [δc 133.6 (C-1 "), 127.4 (C-1 '), 105.3 (C-10)], 8 olefin methine carbon signals [δc 120.6 (C-6"), 115.5 (C-5 "), 111.3 (C -2 "), 105.5 (C-3), 104.7 (C-2 ', 6'), 100.1 (C-6), 95.1 (C-8)] were observed to confirm the presence of a number of benzene rings. In addition, two methoxide carbon signals [δ c 87.4 (C-8 ″), 74.1 (C-7 ″)] and one methylene oxide carbon signal [δ c 61.8 (C-9 ″)] and three methoxy carbon signals [ (δc 56.8 (C-3 '), (C-5'), and 56.3 (C-3 “)] were synthesized, and the results showed that one molecule of flavonoids and one molecule of phenylpropanoids bound together. Determined by bonorigan structure. Linkages of phenylpropanoids H-7 ", 8" and 9 "were identified via COSY spectra in 2D-NMR, and H-7" is C-1 ", 2 from HMBC spectrum. As a result of structural identification by showing a cross peak with "6", the flavonolignan compound represented by the formula (1) was structurally determined by Salcorin B having a phenylpropanoid bonded to a flavonoid compound.

(2) Salmon  C ( salcolin  C)

[Formula 2]

1) yellow powder

2) FAB / MS m / z : 539 [M ⁻ H] ⁻ ; ¹ H-NMR (400 MHz, CD ₃ OD); 7.07 (H-2 ', 6', s), 6.58 (H-3), 6.39 (H-8), 6.13 (H-6), 6.89 (H-2 ", s), 6.73 (H-5" , d, J = 8.0 Hz), 6.81 (H-6 ", dd, J = 8.4 Hz), 4.45 (H-8", d, J = 6 Hz), 3.93 (H-9 "a, dd, J = 12.0, 4.8 Hz), 3.73 (H-9 "b, dd, J = 12.0, 2.6 Hz), 3.29 (H-3 ', H-5', H-7", s), 3.24 (H-3 ", s); ¹³ C-NMR (100 MHz, CD ₃ OD); 183.4 (C-4), 165.9 (C-7), 164.8 (C-2), 162.9 (C-5), 159.1 (C-9), 154.5 (C-3 ', C-5'), 148.6 ( C-3 "), 147.1 (C-4"), 141.4 (C-4 '), 130.9 (C-1 "), 127.3 (C-1'), 105.3 (C-10), 121.9 (C-6 "), 115.5 (C-5"), 111.9 (C-2 "), 105.5 (C-3), 104.7 (C-2 ', 6'), 100.1 (C-6), 95.1 (C-8) , 86.62 (C-8 "), 83.74 (C-7"), 61.7 (C-9 "), 56.7 (C-3 ', C-5'), 56.3 (C-3").

3) Analysis result

Salcorin C isolated in Example 3 was observed to develop on TLC, which showed UV absorption and yellow when sprayed with 10% sulfuric acid and heated. A peak of m / z 539 [MH] ⁻ was observed in FAB / MS, so the molecular weight was determined to be 540 [M + 1] ⁺ . 5 singlet olefin methine proton signals observed in the low field of the ¹ H-NMR (400 MHz, CD ₃ OD, δ _H ) spectrum [δ _H 7.07 (H-2 ′, 6 ′, s), 6.58 (H -3), 6.39 (H-8), 6.13 (H-6)] showed that many benzene rings exist. In addition, two doublet olefin methine proton signals [δ _H 6.89 (H-2 ”, s), 6.73 (H-5”, d, J = 8.0 Hz)] and one doublet olefin methine proton signal [δ _H 6.81 (H-6 ", dd, J = 8.4 Hz)] and the presence of 1,2,4-3 substituted benzene rings. In the region where oxygen was substituted, one methine oxide proton signal [δ _H 4.45 (H-8) ", d, J = 6 Hz)] and one methylene oxide proton signal [δ _H 3.93 (H-9" a, dd, J = 12.0, 4.8 Hz), 3.73 (H-9 "b, dd, J = 12.0, 2.6 Hz)], and three methyl oxide proton signals [δ _H 3.29 (H-3 ′, 5 ′, H-7 ″ s), 3.24 (H-3 ″, s)]. ^{In the 13} C-NMR (100 MHz, CD ₃ OD, δc) spectrum, 24 signals other than 3 methoxy were observed, and DEPT experiments determined the multiplicity of each carbon. Conjugated ketones (δc 183.4) and nine olefin oxide quaternary carbon signals in the low-magnetic region [δc 165.9 (C-7), 164.8 (C-2), 162.9 (C-5), 159.1 (C-9), 154.5 (C-3 ', C-5'), 148.6 (C-3 "), 147.1 (C-4"), 141.4 (C-4 ')] were observed and three olefin quaternary carbon signals [δc 130.9 (C-1 "), 127.3 (C-1 '), 105.3 (C-10)], eight olefin methine carbon signals [δ c 121.9 (C-6"), 115.5 (C-5 "), 111.9 (C -2 "), 105.5 (C-3), 104.7 (C-2 ', 6'), 100.1 (C-6), 95.1 (C-8)] were observed to confirm the presence of a number of benzene rings. In addition, two methoxide carbon signals [δc 86.6 (C-8 ″), 83.7 (C-7 ″)] and one methylene oxide signal [δc 61.7 (C-9 ″)] and two methoxy carbon signals ( δc 56.7, 56.3) were synthesized and determined by the flavonoligan structure in which 1 molecule of phenylpropanoid was bound to 1 molecule of flavonoids similar to Salmonine B. 2D-NMR experiments showed that phenyl was determined by COSY spectrum. Propanoid H-7 ″, 8 ″ and 9 ″ connections were identified and methyl proton oxide (H-7 ″) was determined from the HMBC spectrum by methine oxide (C-1 ″, C-2 ″, C-6 ″). ) And a cross peak with H-7 "showing a linkage with methyl oxide [δc 56.7 (OCH ₃ )], and it was confirmed that OCH ₃ was bonded to C-7". In the case of flavonoids, the spectral data was compared with the literature and determined with tricine. Phenylpropaneoids are derived from trisine C-4 as methine protons (H-8 ") identify the link between 141.4 (C-4") of tricin to determine the linkage between flavonoids and phenylpropanoids. It was confirmed that it is bound to “. As a result of the synthesis, the flavonolignan compound represented by Formula 2 was structurally determined by Salcorin C.

(3) Salcorinoside  A ( salcolinoside  A)

(3)

1) yellow powder

2) FAB / MS m / z : 539 [M ⁻ H] ⁻ ; ¹ H-NMR (400 MHz, CD ₃ OD); 7.07 (H-2 ', 6', s), 6.58 (H-3), 6.39 (H-8), 6.13 (H-6), 6.89 (H-2 ", s), 6.73 (H-5" , d, J = 8.0 Hz), 6.81 (H-6 ", dd, J = 8.4 Hz), 4.45 (H-8", d, J = 6 Hz), 3.93 (H-9 "a, dd, J = 12.0, 4.8 Hz), 3.73 (H-9 "b, dd, J = 12.0, 2.6 Hz), 3.29 (H-3 ', H-5', H-7", s), 3.24 (H-3 ", s); ¹³ C-NMR (100 MHz, CD ₃ OD); 183.4 (C-4), 165.9 (C-7), 164.8 (C-2), 162.9 (C-5), 159.1 (C-9), 154.5 (C-3 ', C-5'), 148.6 ( C-3 "), 147.1 (C-4"), 141.4 (C-4 '), 130.9 (C-1 "), 127.3 (C-1'), 105.3 (C-10), 121.9 (C-6 "), 115.5 (C-5"), 111.9 (C-2 "), 105.5 (C-3), 104.7 (C-2 ', 6'), 100.1 (C-6), 95.1 (C-8) , 86.62 (C-8 "), 83.74 (C-7"), 61.7 (C-9 "), 56.7 (C-3 ', C-5'), 56.3 (C-3").

3) Analysis result

Salcorinoside A isolated in Example 3 was observed to develop on TLC, which showed UV absorption and yellow when sprayed with 10% sulfuric acid and heated. A peak of m / z 687 [MH]? Was observed in the negative FAB-MS, and the molecular weight was determined as 688 [M + 1] ⁺ . In the IR spectrum, the presence of hydroxyl groups (3405 cm ^-1 ) and double bonds (1654 cm ^-1 ) was confirmed. Five olefin methine proton signals observed in the low-field region in the ¹ H-NMR (400 MHz, CD ₃ OD, δ _H ) spectrum [δ _H 7.13 (H-2 ', 6', s), 6.61 (H-3 ), 6.39 (H-8), 6.14 (H-6)] showed that many benzene rings exist. In addition, two doublet olefin methine proton signals (δ _H 7.09 (H-2 ", d, J = 1.2 Hz), δ _H 6.76 (H-5", d, J = 8.0 Hz)] and one doublet olefin From the methine proton signal [δ _H 6.94 (H-6 ", dd, J = 8.0, 1.6 Hz)], the presence of 1,2,4-3 substituted benzene rings was observed. Methine proton signal [δ _H 4.51 (H-8 ”, m)] and one methylene oxide proton signal [δ _H 3.69 (H-9” a, dd, J = 12.0, 4.0 Hz), 3.73 (H-9 ” b, dd, J = 12.0, 2.4 Hz)], three methyl oxide proton signals [δ _H 3.19 (H-3 ′, 5 ′), 5.18 (H-7 ”, d, J = 6.0 Hz), 3.85 ( H-3 ", s)]. In addition, an anomeric proton signal (δ _H 4.62 (H-1 "', d, J = 7.6 Hz)) and methine and methylene proton signals (δ _H 3.8 to 3.3) derived from sugar were observed. From the point of view, it was determined by the structure of the sugar-bonded structure, and was determined by β binding from the anomeric proton coupling constant ( J = 7.6 Hz) ¹³ C-NMR (100 MHz, CD ₃ OD, δc) Spectrum In addition to 3 methoxy, 30 signals were observed, and DEPT experiments were used to determine the multiplicity of each carbon: conjugated ketone (δc 183.4) and 9 olefin oxide quaternary carbon signals [δc 166.1 (C -7), 164.6 (C-2), 162.9 (C-5), 159.1 (C-9), 154.4 (C-3 ', 5'), 148.3 (C-3 "), 147.0 (C-4" ), 140.2 (C-4 ')], three olefin quaternary carbons (δc 131.5 (C-1 "), 127.7 (C-1'), 105.2 (C-10)], eight olefin methines Carbon signals [δc 121.2 (C-6 ″), 115.5 (C-5 ″), 112.4 (C-2 ″), 105.7 (C-3), 104.8 (C-2 ′, 6 ′), 100.2 (C-6), 95.1 (C-8)] were observed to confirm the presence of a number of benzene rings and two methoxide carbon signals [δc 86.8 (C-8 ″), 81.8 (C-7 ″) ] And one methylene oxide carbon signal [δc 61.6 (C-9 ″)] and three methoxy carbon signals [δc 56.9 (C-3 ′, C-5 ′), 56.4 (C-3 ″)] And four methoxide carbon signals derived from anomer carbon [δc 105.2 (C-1 ″ ′)] and sugar [δc 78.0 (C-3 ″ ′), 77.7 (C-5 ″ ′), 75.6 ( C-2 '"), 71.3 (C-4'")] and one methylene oxide carbon signal [δc 62.5 (C-6 '")] are molecules that have been glucopyranose from chemical shifts. Confirmed that β is bound. From the above data, the flavonolignan compound represented by Formula 3 can be expected to have a structure in which β -glucopyranose is bonded to the flavonolignan compound represented by Formula 1. The binding site of sugar was determined by HMBC spectrum, 2D-NMR. In the HMBC spectrum, the anomer carbon signal [δc 105.7 (C-1 ″ ′)] shows a cross peak with a methine oxide proton signal [δ _H 5.18 (H-7 ″, d, J = 6.0 Hz)]. The flavonolignan compound represented by was identified as a structure in which β -glucopyranose is bonded to the C-7 ″ position of salcorin B, and as a result, the flavonolignan compound represented by the formula (3) is Salcorinoside A The structure was determined.

(4) Salcorinoside  B ( salcolinoside  B)

[Formula 4]

1) yellow compound

2) [α] ²⁰ _D- 20 ° ( c = 0.25, MeOH); Negative FAB / MS m / z : 687 [MH] ?; IR (CaF ₂ window, cm ⁻¹ ); 3406, 1652, 1615, 1496, 1456, 1360, 1251, 1123; ¹ H-NMR (400 MHz, CD ₃ OD); 7.04 (H-2 ', 6', s), 6.54 (H-3, s), 6.34 (H-8, s), 6.11 (H-6, s), 7.00 (H-2 ", br s) , 6.73 (H-5 ", d, J = 8.0 Hz), 6.81 (H-6", br d, J = 7.6 Hz), 4.94 (H-7 "), 4.51 (H-8", m), 3.97 (H-9 "), 3.86 (H-3 ', 5', s), 3.81 (H-3", s), 4.33 (H-1 "', d, J = 7.6), 3.99 (H- 9 "a, dd, J = 12.0, 5.2 Hz), 3.68 (H-9" b, dd, J = 12.0, 5.6 Hz), ¹³ C-NMR (100 MHz, CD ₃ OD); 183.3 (C-4), 166.0 (C-7), 164.6 (C-2), 162.8 (C-5), 159.0 (C-9), 154.3 (C-3 ', 5'), 148.4 (C- 3 "), 146.7 (C-4"), 140.4 (C-4 '), 133.2 (C-1 "), 127.2 (C-1'), 105.2 (C-10), 120.8 (C-6") , 115.5 (C-5 "), 111.5 (C-2"), 105.4 (C-3), 104.8 (C-2 ', 6'), 100.2 (C-6), 95.1 (C-8), 86.0 (C-8 "), 74.1 (C-7"), 69.8 (C-9 "), 104.6 (C-1"'), 77.8 (C-3 "'), 77.7 (C-5"'), 75.1 (C-2 "'), 71.5 (C-4"'), 62.6 (C-6 "'), 56.8 (C-3', C-5 '), 56.4 (C-3").

3) Analysis result

Salcorinoside B isolated in Example 3 was observed to develop on TLC, which showed UV absorption and yellow when sprayed with 10% sulfuric acid and heated. The peak of m / z 687 [MH]? was observed in negative FAB-MS, and the molecular weight was determined to be 688 [M + 1] ⁺ . The presence of hydroxyl groups (3406 cm ^-1 ) and double bonds (1652 cm ^-1 ) was confirmed in the IR spectrum. Five olefin methine proton signals observed in the low-field region in the ¹ H-NMR (400 MHz, CD ₃ OD, δ _H ) spectrum [δ _H 7.04 (H-2 ', 6', s), 6.54 (H-3 , s), 6.34 (H-8, s), 6.11 (H-6, s)]. Three olefin methine proton signals [δ _H 7.00 (H-2 ", br s), 6.73 (H-5", d, J = 8.0 Hz), 6.81 (H-6 ", br d, J = 7.6 Hz )], The presence of 1,2,4-3 substituted benzene rings in the oxygen-substituted region, two methine oxide proton signals [δ _H 4.94 (H-7 "), 4.51 (H-8", m )] And one methylene oxide proton signal [δ _H 3.97 (H-9 ")] and two methyl oxide proton signals [δ _H 3.86 (H-3 ', 5', s), 3.81 (H-3", s)], and the anomer proton signal and the methine oxide and methylene proton signals (δ _H 4.3 to 3.9) derived from sugar were observed at δ _H 4.33 (H-1 "', d, J = 7.6). From the point of view, it was expected that the sugar bound structure, it was confirmed that the β bond from the anomeric proton binding constant ( J = 7.6 Hz). ^{In the 13} C-NMR (100 MHz, CD ₃ OD, δc) spectrum, 30 signals other than 3 methoxy were observed, and DEPT experiments determined the multiplicity of each carbon. Conjugated ketone (δc 183.3) and nine olefin oxide quaternary carbon signals in the low-field region [δc 166.0 (C-7), 164.6 (C-2), 162.8 (C-5), 159.0 (C-9), 154.3 (C-3 ', C-5'), 148.4 (C-3 "), 146.7 (C-4"), 140.4 (C-4 ')] were observed and three olefin quaternary carbon signals [δc 133.2 (C-1 "), 127.2 (C-1 '), 105.2 (C-10)], 8 olefin methine carbon signals [δc 120.8 (C-6"), 115.5 (C-5 "), 111.5 (C -2 "), 105.4 (C-3), 104.8 (C-2 ', 6'), 100.2 (C-6), 95.1 (C-8)] were observed to confirm the presence of a number of benzene rings. 2 methoxide carbon signals [δc 86.0 (C-8 ″), 74.1 (C-7 ″)] and 1 methylene oxide signal [δc 69.8 (C-9 ″)], 3 methoxy carbon signals [( δc 56.8 (C-3 ', C-5,'), 56.4 (C-3 ")] were observed, and four of the anomer carbon signal [δc 104.6 (C-1"')] and sugar-derived Methine oxide carbon signal [δc 77.8 (C-3 '"), 77.7 (C-5'"), 75.1 (C-2 '"), 71.5 (C-4'")] and one methylene oxide carbon signal [ delta c 62. 6 (C-6 '") ] of the glucopyranose molecules from the chemical shift was confirmed that the bonded β Bono Plastic From the above data, shown by the general formula 4 lignan compounds to buy Cor B β -. Glucopyranose The binding site of sugar was determined by HMBC spectrum, which is 2D-NMR, in which the anomer carbon signal [δc 104.6 (C-1 ′ ′)] was methylene proton signal [δ _H 3.97 (H-9 ")] and the flavonolignan compound represented by the formula (4) showing a cross peak was identified as a structure in which β -glucopyranose is bonded to the C-9" position of Salcorin B, and synthesized as a result The flavonolignan compound represented by the formula (4) was structurally determined by Salcorinoside B.

(5) Salcorinoside  C ( salcolinoside  C)

[Chemical Formula 5]

1) yellow powder

2) Negative FAB / MS m / z : 687 [MH] ?; IR (KBr, MeOH, cm-1); 3405, 1654, 1616, 1496, 1358, 1252, 1165, 1123; ¹ H-NMR (400 MHz, CD ₃ OD); (H-2 ', 6', brs), 6.62 (H-3), 6.40 (H-8), 6.16 , d, J = 8.0 Hz) , 6.91 (H-6 ", dd, J = 8.4, 1.6 Hz), 5.28 (H-7", d, J = 4.0 Hz), 4.43 (H-8 ", m) , 4.18 (1H, d, J = 6.8 Hz), 3.83 (H-3 ', H-5' , J = 12.0, 6.4 Hz) , 3.68 (H-9 "b, dd, J = 12.0, 6.0 Hz), 13 C-NMR (100 MHz, CD 3 OD); 183.4 (C-4), 166.0 (C C-4), 164.9 (C-2), 162.9 (C-5), 159.1 ), 115.4 (C-5), 112.8 (C-12), 140.7 (C-4 '), 130.4 (C-2), 105.6 (C-3), 104.6 (C-2 ', 6'), 100.1 (C-3), 77.7 (C-5 "'), 75.0 (C-2"'), 71.8 (C-4 '''), 62.7 (C-6'''), 56.3 (C-3 ', C-5'), 49.6 (C-3 '').

3) Analysis result

Salcorinoside C isolated in Example 3 was observed to develop on TLC, which showed UV absorption and yellow when sprayed with 10% sulfuric acid and heated. A peak of m / z 687 [MH]? Was observed in the negative FAB-MS, and the molecular weight was determined as 688 [M + 1] ⁺ . In the IR spectrum the presence of hydroxyl (3405 cm ⁻¹ ) and double bond (1654 cm ⁻¹ ) was confirmed. Five olefin methine proton signals observed in the low-field region in the ¹ H-NMR (400 MHz, CD ₃ OD, δ _H ) spectrum [δ _H 7.19 (H-2 ', 6', brs), 6.62 (H-3 ), 6.40 (H-8), 6.16 (H-6)] showed that many benzene rings exist. In addition, two doublet olefin methine proton signals [δ _H 7.10 (H-2 ”, brs), δ _H 6.76 (H-5”, d, J = 8.0 Hz) and one doublet olefin methine proton signals [δ _H 6.91 (H-6 ", dd, J = 8.4, 1.6 Hz)] confirmed the presence of 1,2,4-3 substituted benzene rings. In the oxygen substituted region, one methine oxide proton signal [δ _H 4.43 (H-8 ", m)] and one methylene oxide proton signal [δ _H 3.92 (H-9" a, dd, J = 12.0, 6.4 Hz), 3.68 (H-9 "b, dd, J = 12.0, 6.0 Hz)], three methyl oxide proton signals [δ _H 3.83 (H-3 ', H-5', s), 5.28 (H-7 ", d, J = 4.0 Hz), 3.79 (H- 3 ", s)]. The anomer proton signal [δ _H 4.18 (H-1 ”, d, J = 6.8 Hz)] and the methine and methylene proton signals (δ _H 3.8 to 3.3) derived from sugar were observed. Structure was estimated and determined by α binding from anomer proton binding constant ( J = 6.8 Hz) In the ¹³ C-NMR (100 MHz, CD ₃ OD, δc) spectrum, 30 signals were observed in addition to 3 methoxy. In the low field, the conjugated ketone (δc 183.4) and nine olefin oxide quaternary carbon signals [δc 166.0 (C-7), 164.9 (C-2), 162.9 were determined. (C-5), 159.1 (C-9), 154.4 (C-3 ', C-5'), 148.5 (C-3 "), 147.0 (C-4"), 140.7 (C-4 ')] 3 olefin quaternary carbon signals [δ c 130.4 (C-1 ″), 127.5 (C-1 ′), 105.2 (C-10)], 8 olefin methine carbon signals [δ c 121.9 (C-6) "), 115.4 (C-5"), 112.8 (C-2 "), 105.6 (C-3), 104.6 (C-2 ', 6'), 100.1 (C-6), 95.1 (C-8) ] Is observed The presence of a number of benzene rings was confirmed, two methoxide carbon signals [δ c 86.8 (C-8 ″), 77.8 (C-7 ″)] and one methylene oxide carbon signal [δ c 61.8 (C-9 ″) ], Three methoxy carbon signals [δc 56.3 (C-3 ′, C-5 ′), 49.6 (C-3 ″)] and anomer carbon [δc 100.7 (C-1 ″ ′)]. Four methoxide carbon signals derived from fructose [δc 77.8 (C-3 "'), 77.7 (C-5"'), 75.0 (C-2 "'), 71.8 (C-4"') and It was confirmed from the chemical shift of one methylene oxide carbon signal [δc 62.7 (C-6 ′ ″)] that one molecule of glucopyranose was α- bonded. From the above data, the flavonolignan compound represented by Formula 5 Expected to be a structure in which glucopyranose is bound to Corin B. The binding site of sugar was determined by HMBC spectrum, 2D-NMR. In the HMBC spectrum, the anomer carbon signal [δc 100.7 (C-1 ″ ′)] shows a cross peak with a methine oxide proton signal [δ 5.28 (H-7 ″, d, J = 4.0 Hz)] to the formula (5). The flavonolignan compound to be displayed was identified as a structure in which glucopyranose α was bonded to the C-7 ″ position of salcorin B. As a result, the flavonolignan compound represented by the formula (5) was determined as salcorinoside C. It was.

Experimental Example  One: Flavono League  Determination of anti-inflammatory activity of compounds

(1) cell culture

RAW 264.7 cells (Korean Cell Line Bank; KCLB ) were cultured in DMEM medium containing 10% FBS and penicillin (100 μg / mL), streptomycin (100 U / mL). C was incubated in a 5% CO ₂ incubator. 1 hour pretreatment of RAW 264.7 cells with different concentrations (0, 1.56, 3.125, 6.25, 12.5, 25, 50, 100 μM) or a positive control of the sample solution containing the flavonolignan compound represented by Formula 1 to Formula 5 After treatment with LPS (1 μg / mL) and incubated for 24 hours.

(2) cytotoxicity test

The cells were equally divided into 1 × 10 ⁵ cells / well in a 96-well plate and incubated for 24 hours, and then sample solutions of various concentrations were added in two groups. After 1 hour, only one group was treated with LPS (1 μg / mL). After 24 hours, MTT reagent was added and left for 4 hours. The supernatant was removed and the formed formazan was dissolved by adding 100 μL of DMSO. After 30 minutes the absorbance was measured at 540 nm. The survival rate of the cells was measured by the ratio of the viable cells in the group treated with the flavonolignan compound represented by Formula 1 to Formula 5 and the control group treated with nothing according to the following Equation 1, and the results are respectively shown in FIG. To FIG. 5.

[Equation 1]

Cell survival rate (%) = [OD (compound treated cell number)-OD (blank cell number) / OD (control cell number)-OD (blank cell number)] × 100

(3) nitrogen monoxide ( Nitric Oxide , NO A measure of quantity

The amount of NO generated from RAW 264.7 cells was measured in the form of NO ₂ ⁻ present in the cell culture using Griess reagent. 100 μL of cell culture supernatant and 100 μL of Griess reagent [1% (w / v) sulfanilamide in 5% (v / v) phosphoric acid and 0.1% (w / v) naphtylethylenediamine-HCl] were mixed in a 96-well plate. After reacting for 10 minutes, absorbance was measured at 540 nm. Cell culture supernatants were treated with RAW 264.7 cells at different concentrations (0, 1.56, 3.125, 6.25, 12.5, 25, 50, 100 μM) of the sample solution containing the flavonolignan compound represented by Formulas 1 to 5 above. As a result, the amount of nitrogen monoxide was measured for five kinds of cell culture supernatants, and the results are shown in FIGS. 6 to 10, respectively.

(4) results

1 to 10, in the case of Salcorin B represented by the formula (1), it was confirmed the concentration-dependent inflammation inhibitory activity. In particular, at a concentration of 25 μM showed no cytotoxic effect, 50% NO production inhibitory activity was confirmed.

In the case of Salcorin C represented by the formula (2), it was confirmed a very good inflammatory inhibitory activity in a concentration-dependent, the NO production inhibitory activity of about 60% was confirmed without a cytotoxic effect at a concentration of 12.5 μM. However, the concentration of 100 μM showed cytotoxicity of RAW 264.7.

In the case of Salcorinoside A represented by the formula (3), the inhibitory activity was confirmed in a concentration-dependent manner, at a concentration of 50 μM showed no cytotoxic effect, but confirmed the NO production inhibitory activity of about 40%, the cells of RAW 264.7 No toxicity was confirmed.

In the case of salcorinoside B represented by the formula (4), it was confirmed that the concentration-dependent inflammation inhibitory activity, the NO production inhibitory activity of about 80% without showing a cytotoxic effect at a concentration of 100 μM.

In the case of salcorinoside C represented by the formula (5), it was confirmed that the inflammation inhibitory activity in a concentration-dependent manner, the NO production inhibitory activity of about 80% without showing a cytotoxic effect at a concentration of 100 μM.

Claims (13)

A pharmaceutical composition for the prevention and treatment of inflammatory diseases, containing rice ground extract as an active ingredient.
The method of claim 1,
The ground rice extract is a pharmaceutical composition for the prevention and treatment of inflammatory diseases, characterized in that the extract obtained by extracting the ground rice body biologically with an aqueous alcohol solution.
The method of claim 2,
The aqueous alcohol solution is a pharmaceutical composition for the prevention and treatment of inflammatory diseases, characterized in that 80% aqueous methanol solution.
A pharmaceutical composition for the prevention and treatment of inflammatory diseases, comprising the rice ground fraction obtained by fractionation from the above-ground rice extract of claim 2 as an active ingredient.
The method of claim 4, wherein
The rice plants above-ground fraction of rice shoot extract n -, characterized in that the obtained extract partitioned between butanol-extract partitioned between hexane and H ₂ O and the extracted allocation of the H ₂ O layer with ethyl acetate, H ₂ O layer n Pharmaceutical compositions for the prevention and treatment of inflammatory diseases.
A pharmaceutical composition for the prophylaxis and treatment of inflammatory diseases, which comprises a flavonolignan compound or a pharmaceutically acceptable salt thereof obtained as separated from the above-ground rice fraction of claim 5 as an active ingredient.
The method of claim 6,
The flavonolignan compound is a pharmaceutical composition for the prevention and treatment of inflammatory diseases, characterized in that salcolin B (Salcolin B) represented by the formula (1):
[Formula 1]

.
The method of claim 6,
The flavonolignan compound is a pharmaceutical composition for the prevention and treatment of inflammatory diseases, characterized in that salcolin C represented by the formula (2):
(2)

.
The method of claim 6,
The flavonolignan compound is a pharmaceutical composition for the prevention and treatment of inflammatory diseases, characterized in that salcolinoside A (Salcolinoside A) represented by the formula (3):
(3)

.
The method of claim 6,
The flavonolignan compound is a pharmaceutical composition for the prevention and treatment of inflammatory diseases, characterized in that salcolinoside B (Salcolinoside B) represented by the formula (4):
[Chemical Formula 4]

.
The method of claim 6,
The flavonolignan compound is a pharmaceutical composition for the prevention and treatment of inflammatory diseases, characterized in that salcolinoside C (salcolinoside C) represented by the formula (5):
[Chemical Formula 5]

.
The method according to any one of claims 1, 4 and 6,
The inflammatory disease is rhinitis, bronchitis, arthritis, sepsis, periodontitis, gastric ulcer, pancreatitis, inflammatory skin disease or inflammatory bowel disease, characterized in that the pharmaceutical composition for the prevention and treatment of inflammatory diseases.
The method according to any one of claims 1, 4 and 6,
The pharmaceutical composition is a pharmaceutical composition for preventing and treating inflammatory diseases, characterized in that it has an NO production inhibitory activity from the cells.

Images