KR101800785B1 - A composition comprising compounds isolated from Echinochloa utilis for preventing or treating inflammatory disease - Google Patents

Abstract

The present invention relates to a composition for preventing or treating an inflammatory disease comprising a compound isolated from edible fatigue. The compound has excellent NO activity inhibitory activity and can be usefully used as a composition for preventing or treating an inflammatory disease.

Description

[0001] The present invention relates to a composition for preventing or treating an inflammatory disease,

The present invention relates to a composition for the prophylaxis or treatment of inflammatory diseases comprising a compound isolated from edible fatigue.

Inflammation (inflammation) is caused by damage to tissues (cells) or infection with external infectious agents (bacteria, fungi, viruses, various allergens), various inflammatory mediators and immune cells are involved, Complex physiological responses such as substance secretion, fluid infiltration, cell migration, and tissue destruction, and external symptoms such as erythema, edema, fever, and pain. In the normal case, the inflammatory reaction removes the external infectious agent and regenerates the damaged tissue to regenerate the organism's function. However, when the antigen is not removed or the internal substance causes the inflammatory reaction to occur excessively or continuously, , And some of them lead to rheumatoid arthritis, osteoporosis, sepsis, vascular diseases, cancer and the like. In other words, the inflammatory response is the most important pathological mechanism of the host defense mechanism against the wound, microbial infection, but persistent and excessive inflammation damages the tissue.

Macrophages are the most representative immune cells that regulate these inflammatory responses. They play an important role in host defense mechanisms in harmful environments and are involved in the progress of various diseases such as autoimmune diseases. Inflammatory mediators such as lipopolysaccharide can activate macrophages and activated macrophages can induce various inflammatory mediators such as TNF-α, IL-6 (interleukin-6) and IL-1β produces and generates iNOS (inducible nitric oxide synthase) and COX-2 (cyclooxygenase-2) to synthesize a NO (nitric oxide) and _{PGE 2 (prostaglandin E 2) (} Jin, HJ et al., 2010).

NO is a physiologically important homeostatic regulator and plays a variety of roles, such as eliminating intestinal bacteria and tumors, regulating blood pressure, or mediating neurotransmission. However, when inflammation occurs, the amount of iNOS expressed in the inflammation-related cells is increased to produce a large amount of NO. Excessively generated NO causes tissue damage, genetic mutation, nerve damage, and increases vascular permeability and induces edema . When an inflammatory reaction occurs, COX-2 is produced by macrophage PGE ₂ , which is an inflammatory factor mainly responsible for pain and fever (Wang, MT et al., 2007). Therefore, the anti-inflammatory effect can be confirmed by confirming the inhibition of the production of NO and COX-2 produced in the inflammatory reaction.

Recently, it has been reported that foods and plants have the function of preventing or treating diseases. In order to meet the needs of humans who want to live healthier and longer lives, researches to search for substances having diverse physiological functions from various resources worldwide (Lee, SE et al., 2004). In particular, plant-based compounds have been attracting much attention.

Edible blooms ( Echinochloa utilis, Barnyard millet) are cultivated mainly in Korea, Japan, and northeastern China. Edible blooms are highly adaptable to the environment, so they can grow in mountains, barren lands, and high salinity areas, making them promising as decontamination crops for reclaimed land and clearing land, as well as a relatively short growing period of 90 to 110 days. Available. In addition, it has good climate tolerance, low water requirement, good climate adaptability, C4 biomass is available as feed crops and bio-energy crops (Lee Seong-gap, 2009).

It is also known that edible blood has a higher content of nutrients such as essential amino acids and unsaturated fatty acids than other grains, and is known to contain a large amount of physiologically active substances having antioxidative activity. Thus, (Kim, JY et al., 2011). It has been reported that it can be used as a health food.

As a prior art related to edible blood, Korean Patent No. 10-1222779 discloses a pharmaceutical composition for preventing or treating diabetes comprising edible blood extract, Korean Patent Laid-Open No. 10-2013-0051180 It has been disclosed with respect to a whitening cosmetic composition comprising an edible bark extract and a compound separated therefrom, but it has been disclosed yet that the compound of the present invention isolated from edible fatigue can be used for the prophylactic or therapeutic use of inflammatory diseases none.

Korean Patent Laid-Open No. 10-2013-0051180, a cosmetic composition for whitening comprising an edible blood extract and a compound isolated therefrom, disclosed on May 20, 2013. Korean Patent No. 10-1222779, Pharmaceutical composition for prevention and treatment of diabetes comprising edible blood extract as an active ingredient, registered on Jan. 09, 2013.

Lee Sung - Gab, production processing and utilization of granulated pork in China, 2009. Jin, H. J. et al., Anti-inflammatory and anti-arthritic activity of total flavonoids of the roots of Sophora flavescens, J. Ethnopharmacol., 127 (3), 589-595, 2010. Kim, J. Y. et al., Physicochemical and antioxidative properties of selected barnyard millet (Echinochloa utilis) species in Korea, Food Sci Biotechnol., 20, 461-469, 2011. Lee, S. E. et al., Inhibitory activity of angiotensin converting enzyme (ACE) in Korean medical herbs, J Medicinal Crop Sci., 12, 73-78, 2004. Migliorini, P. et al., Macrophage NO2-production as a sensitive and rapid assay for quantitation of murine IFN-gamma, J Immunol Methods., 139 (1), 107-114, Wang, M. T. et al., Cyclooxygenases, prostanoids, and tumor progression, Cancer Metastasis Rev., 26 (3-4), 525-534, 2007.

It is an object of the present invention to provide a composition for preventing or treating an inflammatory disease comprising a compound isolated from edible fatigue, and also to provide a composition for preventing or treating inflammatory diseases, .

The present invention relates to a process for the production of Echinochloa to separate from the utilis) of Formula 1 (±) - anti-1- (4-hydroxy-3-methoxyphenyl) -2- {4 - [(E ) -3- acetoxy-propene-1-one ] -2-methoxyphenoxy} propane-1,3-diol 3-acetate (Compound 1), Skispen lignan I (Compound 2), dimer coniferyl acetate (Compound 3) 4), Lucy denal (compound 5), 9,9'-bis-acetyl-Neo-up Bill (compound 6), (2 S, 3 S, 4 S, 5 S) -3,4- furan dimethanol, tetra (Compound 7), echinoclorin A (compound 8), echinochlorine B (compound 9), 2 (methylhydroxyphenyl) -3,4-diacetate ', 3'-dihydroxypropyl na decanoate (compound 10) and (8 E, 11 E) -2', 3'-dihydroxypropyl octa decanoic -8,11- diethoxy furnace benzoate (compound 11) And a composition for preventing or treating inflammatory diseases, which comprises at least one compound selected from the group consisting of The.

[Chemical Formula 1]

Said inflammatory disease is selected from the group consisting of allergic diseases, inflammatory bowel disease, atherosclerosis, inflammatory collagen vascular disease, glomerulonephritis, inflammatory skin disease, sarcoidosis, retinitis, gastritis, hepatitis, enteritis, arthritis, tonsillitis, sore throat, bronchitis, pneumonia, pancreatitis, Septicemia, and nephritis. ≪ RTI ID = 0.0 >

In addition, the present invention relates to a method for producing Echinochloa wherein in formula (1) is separated from utilis) (±) - anti-1- (4-hydroxy-3-methoxyphenyl) -2- {4 - [(E ) -3- acetoxy-propene-1-one ] -2-methoxyphenoxy} propane-1,3-diol 3-acetate (Compound 1), Skispen lignan I (Compound 2), dimer coniferyl acetate (Compound 3) 4), Lucy denal (compound 5), 9,9'-bis-acetyl-Neo-up Bill (compound 6), (2 S, 3 S, 4 S, 5 S) -3,4- furan dimethanol, tetra (Compound 7), echinoclorin A (compound 8), echinochlorine B (compound 9), 2 (methylhydroxyphenyl) -3,4-diacetate ', 3'-dihydroxypropyl na decanoate (compound 10) and (8 E, 11 E) -2', 3'-dihydroxypropyl octa decanoic -8,11- diethoxy furnace benzoate (compound 11) As an active ingredient, at least one compound selected from the group consisting of: < RTI ID = 0.0 > One will.

In another aspect, the present invention provides a novel compound having a chemical structure represented by the general formula (1), (±) -anti-1- (4-hydroxy-3-methoxyphenyl) -2- {4- [ (Compound 1), echinoclorin A (compound 8), and echinoclorine B (compound 9) were obtained in the same manner as in Example 1, except that 3-acetoxypropen-1-yl] -2-methoxyphenoxy} propane- ).

The present invention also provides a method for producing an edible blood extract, comprising: extracting edible blood with water, C1 to C4 lower alcohol or a mixed solvent thereof to prepare an edible blood extract; Sequentially fractionating the edible extract using n-hexane, methylene chloride, ethyl acetate and n-butanol; And each of the above fractions was subjected to chromatography to obtain (±) -anti-1- (4-hydroxy-3-methoxyphenyl) -2- {4 - [(E) -3- Methoxyphenoxy} propane-1,3-diol 3-acetate (compound 1), squispan lignan I (compound 2), dimer coniferyl acetate (compound 3) siringa Lecinol (compound 4), Lucy denal (compound 5), 9,9'-bis-acetyl-Neo-up Bill (compound 6), (2 S, 3 S, 4 S, 5 S) -3,4- (Compound 7), echinoclorine A (compound 8), echinoclorine B ((meth) acrylate) compound 9), 2 ', 3'-dihydroxypropyl na decanoate (compound 10) and (8 E, 11 E) -2', 3'-dihydroxypropyl octa decanoic -8,11- diethoxy furnace (Compound 11). The present invention also relates to a method for separating at least one compound selected from the group consisting of Compound

Hereinafter, the present invention will be described in detail.

The present invention relates to a composition for the prevention or treatment of inflammatory diseases, which comprises at least one compound selected from the group of compounds of the formula (1) isolated from edible blood ( Echinochloa utilis ).

The compound separated from the edible fatigue can be obtained by fractionating the edible blood by chromatography from water, extracts of C1 to C4 lower alcohols or a mixed solvent thereof, and the C1 to C4 lower alcohols include methanol, ethanol , Propanol, isopropanol, butanol, and the like.

In addition, the chromatography can be carried out by silica gel column chromatography, flash column chromatography, sephadex LH-20 column chromatography, RP-18 column chromatography (RP-18 column chromatography), thin layer chromatography (TLC), medium pressure liquid chromatography and high performance liquid chromatography (HPLC).

Meanwhile, the compound of the present invention can be synthesized according to a conventional method in the art, and can also be prepared as a pharmaceutically acceptable salt.

The present invention also provides a pharmaceutical composition for the prevention or treatment of inflammatory diseases, which comprises at least one compound selected from the group of compounds of the formula (1) separated from edible fatigue. The pharmaceutical composition containing the compound can be formulated in the form of oral, granule, tablet, capsule, suspension, emulsion, syrup, aerosol or the like oral preparation, external preparation, suppository and sterilized injection solution according to a conventional method Can be used. Examples of carriers, excipients and diluents that can be contained in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose , Methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. 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 formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient, such as starch, calcium carbonate, sucrose or lactose, Gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

The dosage of the pharmaceutical composition comprising at least one compound selected from the group of compounds of formula 1 isolated from the edible fat of the present invention will depend on the age, sex, weight, specific disease or condition, disease or pathology to be treated, The severity of the condition, the route of administration, and the judgment of the prescriber. Dosage determinations based on these factors are within the level of ordinary skill in the art and generally the dosage ranges from 0.01 mg / kg / day to approximately 2000 mg / kg / day. A more preferable dosage is 1 mg / kg / day to 500 mg / kg / day. The administration may be carried out once a day or divided into several times. The dose is not intended to limit the scope of the invention in any way.

The pharmaceutical composition comprising at least one compound selected from the group of compounds of formula 1 isolated from the edible fat of the present invention can be administered to mammals such as rats, livestock, and humans by various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine dural or intracerebral injection. Since the compound has little toxicity and side effects, it can be safely used for long-term use for preventive purposes.

The present invention also provides a health functional food for preventing or ameliorating an inflammatory disease comprising at least one compound selected from the group of compounds of the formula (1) isolated from edible fatigue and a pharmaceutically acceptable food-aid additive. The compound may be added in an amount of 0.001 to 100% by weight to the health functional food of the present invention. The health functional food of the present invention includes forms such as tablets, capsules, pills, and liquids, and examples of foods to which the compound of the present invention can be added include various foods, beverages, gums, tea, vitamins .

The present invention relates to a composition for preventing or treating an inflammatory disease comprising a compound isolated from edible fatigue. The compound has excellent NO activity inhibitory activity and can be usefully used as a composition for preventing or treating an inflammatory disease.

1 is a figure showing a correlation (arrow) of ^{1 H- 1 H COSY (bold lines} ) and the ¹ H- ¹³ C HMBC for the novel compounds of Compounds 1, 8 and 9 of the invention.
2 is a graph showing cell viability for the compounds 1 to 11 of the present invention.
FIG. 3 shows the result of measuring the amount of NO produced after applying the compounds 1, 2, 3, 5, 6, 9 and 11 of the present invention to macrophages treated with LPS.
Fig. 4 shows the results of the expression of the proteins iNOS and COX-2, which are proteins involved in inflammation, when LPS-treated macrophages are treated with compounds 1, 2, 3, 5, 6 and 9 of the present invention .

Hereinafter, preferred embodiments of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein but may be embodied in other forms. Rather, the intention is to provide an exhaustive, complete, and complete disclosure of the principles of the invention to those skilled in the art.

≪ Example 1 > Separation of food-derived compounds for edible purposes &

The edible blood ( Echinochloa utilis ) used in the present invention was received in February 2013 from the National Institute of Crop Science.

30 kg of the edible seeds were extracted with 80% ethanol (4 times × 20 liter) at 80 ° C. for 4 hours, and the ethanol extract obtained in the above procedure was concentrated under reduced pressure to obtain 1.1 kg of an ethanol extract. Thereafter, the ethanol extract was suspended in 6 L of water and washed with n-hexane (6 L × 7), methylene chloride (6 L × 7), ethyl acetate (6 L × 7), and n-butanol (6 L × 7) Were sequentially fractionated to obtain a n-hexane fraction (305 g), a methylene chloride fraction (68 g), an ethyl acetate fraction (35 g) and an n-butanol fraction (130 g) and finally a water fraction. The methylene chloride fractions and ethyl acetate fractions, which had comparatively high activity to inhibit NO production in the fractions, and which had similar TLC analysis results, were combined and mixed with n-hexane: ethyl acetate: methanol (1: 0: 0 → 1: 1: 0 (Particle size: 63-200 mu m, column size: 10 x 120 cm) according to concentration gradient elution conditions of 1: 1: 0.1 to 1: 1: And 14 small fractions (A to N) were obtained.

As a result of measuring the NO production inhibitory activity using the 14 small fractions, the small fractions G and the small fractions H showed the strongest inhibitory activity, and the active compounds were separated from the two small fractions.

The small fraction G (2.8 g) was fractionated under the conditions of RP - 18 column chromatography and methanol: water (1: 1, 2: 1, 4: 1, 8: 1, 12: G-2 (98.7 mg) was fractionated by semi - preparative HPLC (RS tech Optima Pak C18 column, column size: 10 × 250 mm) in a 50% methanol aqueous solution for 40 minutes , Particle size: 5 탆) to obtain Compound 5 (6 mg). The small fraction G-3 (183 mg) was eluted under the conditions of silica gel column chromatography (particle size: 63-200 탆, column size: 2.5 횞 50 cm) and methylene chloride: acetone (10: Two small fractions G-3-1 and G-3-2 were obtained, and the small fraction G-3-1 (63 mg) was subjected to semi - preparative HPLC (RS tech Optima Pak C18 column, column size: 10 x 250 mm, particle size: 5 탆) to obtain Compound 6 (6.7 mg) and Compound 7 (16.6 mg). The small fraction G-7 (97 mg) and the small fraction G-8 (25 mg) were separated by Sephadex LH-20 column chromatography (particle size: 25-100 μm, column size 2.5 × 1200 cm) To obtain Compound 3 (37 mg) and Compound 9 (8.2 mg), respectively. Further, the small fraction G-13 (54.5 mg) was fractionated by 30% acetonitrile aqueous solution (6 L) and RP-18 column chromatography (column size: 4.5 x 50 cm) to obtain compound 10 (23 mg) G-14 (180 mg) was fractionated by a 50% acetonitrile aqueous solution (6 L) and RP-18 column chromatography (column size: 4.5 x 50 cm) to obtain Compound 11 (61 mg).

The small fraction H (2.4 g) was fractionated with Sephadex LH - 20 column chromatography (particle size: 25-100 탆, column size 2.5 x 1200 cm) and 100% methanol to obtain five small fractions (H-1 to H H-3 (623 mg) of the above-mentioned small fractions was dissolved in RP-water (1: 1, 2: 1, 4: 1, 8: 18 column chromatography (particle size: 75-140 占 퐉, column size: 5 占 60 cm) to obtain six small fractions (H-3-1 to H-3-6). H-3-2 (386 mg) and H-3-5 (52.1 mg) were fractionated by silica gel column chromatography (particle size: 63-200 μm, column size: 2.5 × 50 cm) and methylene chloride : Ethyl acetate (4: 1 and 6: 1, 3 L, respectively) to obtain Compound 1 (15.0 mg) and Compound 8 (22.0 mg), respectively. The fraction H-4 (323 mg) was fractionated by RP - 18 column chromatography (particle size: 75-140 μm, column size: 2.5 × 60 cm) and methanol: water (3: 2) H-4-3 (203 mg) of the above fractions were subjected to RP-18 column chromatography (column size: 4.5 x 50 cm) and 66% Compound 2 (35.6 mg) was fractionated using an aqueous methanol solution (4 L) and the small fraction H-4-6 (32.1 mg) was purified by RP-18 column chromatography and methanol: water (3: 2) Mg).

<Example 2> Identification of Physico-Chemical Structure of Edible Pigment-Derived Compound>

The physicochemical properties of the edible poultry derived compounds 1 to 11 isolated and obtained in Example 1 are as follows.

Example  2-1. (±) - Anti -1- (4-hydroxy-3- Methoxyphenyl ) -2- {4 - [(E) -3- Acetoxypropene -1-yl] -2-methoxyphenoxy} propane-1,3-diol 3-acetate (Compound 1)

(E) -3-acetoxypropen-1-yl] -2-methoxyphenoxy} propane-1,3-diol 3- {3- [ acetate;

Colorless oil;

0°(c 0.05, MeOH);

0 [deg.] ( C 0.05, MeOH);

UV (MeOH) ? _Max nm (log ? ): 203 (4.23), 259 (3.84);

IR (KBr) ? _Max cm ^-1 : 3666, 3416, 2971, 2337, 1736, 1598, 1510, 1477, 1054;

¹ H NMR and ¹³ C NMR data, Table 1 and Table 2;

HR-FAB-MS m / z 460.1731 (calcd for C ₂₄ H ₂₈ O ₉ ).

Example 2-2. Skispen lignan I (Compound 2)

schisphenlignan I;

Amorphous powder;

-13.5°(c 0.17, MeOH);

-13.5 [deg.] ( C 0.17, MeOH);

_{UV (CHCl 3) λ max (} log ε): 204 (2.73);

IR ? _Max cm ^-1 : 3425, 2923, 2853, 1741, 1616, 1518, 1465, 1240, 1146, 1034;

_{^{1 H NMR (Methanol- d 4,}} 600 MHz) δ: 6.90 (1H, overlapped, H-2 '), 6.92 (1H, br s, H-6'), 6.50 (1H, d, J = 15.8 Hz, H-7 '), 6.19 ( 1H, dt, J = 5.8, 15.8 Hz, H-8'), 4.16 (2H, d, J = 5.8 Hz, H-9 '), 6.78 (1H, dd, J = 1.7, 8.1 Hz, H-2 ), 6.74 (1H, d, J = 8.1 Hz, H-3), 6.90 (1H, overlapped, H-6), 5.41 (1H, d, J = 7.02 Hz, H- 7), 3.68 (1H, m , H-8), 4.28 (1H, dd, J = 5.2, 11.1 Hz, H-9a), 4.37 (1H, dd, J = 7.4, 11.1 Hz, H-9b), _{3.77 (3H, s, OCH 3} ), 3.82 (3H, s, OCH 3), 1.95 (3H, s, OCOCH 3);

_{^{13 C NMR (Methanol- d 4,}} 150 MHz) δ: 133.8 (C-1), 110.7 (C-2 '), 149.3 (C-5), 148.0 (C-4), 116.4 (C-3), 2), 145.8 (C-2), 90.0 (C-7), 51.8 (C-8), 66.8 ), 149.3 (C-4 '), 129.4 (C-3'), 112.5 (C-6 '), 132.0 _{20.8 (OCOCH 3), 56.6 (} OCH 3), 56.9 (OCH 3), 172.8 (OCOCH 3);

HR-ESI-MS m / z [M + Na] + 423.1424 (calcd. For C 22 H 24 O 7).

Examples 2-3. Dimer coniferyl acetate (Compound 3)

dimeric coniferyl acetate;

Colorless oil;

-2.0°(c 1.2, CH₂Cl₂);

_{-2.0 ° (c 1.2, CH 2} Cl 2);

_{UV (CHCl 3) λ max ㎚} (log ε): 279 (4.19), 304 (3.86), 347 (3.29);

IR ? _Max cm ^-1 : 3460, 3020, 2955, 2855, 1740, 1732, 1604, 1516, 1500, 1464, 1432, 1380, 1364, 1332, 1250, 1146, 1034, 960, 928, 856, 734;

_{^{1 H NMR (Methanol- d 4,}} 600 MHz) δ: 6.90 (1H, overlapped, H-2 '), 6.93 (1H, br s, H-6'), 6.56 (1H, d, J = 15.8 Hz, H-7 '), 6.15 ( 1H, dt, J = 5.8, 15.8 Hz, H-8'), 4.64 (2H, d, J = 6.5 Hz, H-9 '), 6.78 (1H, dd, J = (1H, d, J = 7.08 Hz, H-2), 6.74 (1H, d, J = 8.2 Hz, H- 7), 3.68 (1H, m , H-8), 4.27 (1H, dd, J = 7.5, 11.1 Hz, H-9a), H-9b 4.37 (1H, dd, J = 5.1, 11.1 Hz,), _{3.77 (3H, s, OCH 3} ), 3.82 (3H, s, OCH 3), 2.01 (3H, s, OCOCH 3), 1.95 (3H, s, OCOCH 3);

_{^{13 C NMR (Methanol- d 4,}} 150 MHz) δ: 133.7 (C-1), 116.7 (C-2 '), 149.8 (C-5), 148.1 (C-4), 116.4 (C-3), 2), 145.8 (C-2), 90.0 (C-7), 51.8 (C-8), 66.7 ), 149.3 (C-4 '), 129.5 (C-3'), 112.6 (C-6 '), 135.6 _{20.8 (OCOCH 3), 56.6 (} OCH 3), 56.9 (OCH 3), 172.9 (OCOCH 3), 172.7 (OCOCH 3);

ESI-MS m / z [MH] ^- 441 (calcd for C ₂₄ H ₂₆ O ₈ ).

Examples 2-4. (+) Cyringaredinol (Compound 4)

(+) syringaresinol;

Yellow powder;

+42°(c 0.1, CHCl₃);

+ 42 ° (c 0.1, CHCl 3);

UV (MeOH) ? _Max nm (log ? ): 240 (4.08), 271 (3.88);

IR ? _Max cm ^-1 : 3340, 1610, 1521, 1456, 1425, 1377, 1319;

^{_{1 H NMR (Methanol- d 4,}} 600 MHz) δ: 6.60 (4H, s, H-2, 6, 2 ', 6'), 4.67 (2H, d, J = 3.6 Hz, H-7, 7 ' ), 4.21 (2H, dd, J = 6.0, 9.0 Hz, H-9a, 9'a), 3.85 (2H, dd, J = 6.8, 9.0 Hz, H- m), 3.79 (12H, s , 4 'OCH 3);

¹³ C NMR (Methanol- d ₄ , 150 MHz) ?: 131.1 (C-1,1 '), 104.7 (C-2, 2', 6, 6 ' 5 '), 136.4 (C- 4, 4'), 87.8 (C-7, 7 '), 55.7 (C-8, 8'), 72.9 (C-9, 9 '), 57.0 (OCH 3);

EI-MS m / z [M ] + 418 (calcd. For C 22 H 26 O 8).

Examples 2-5. Lucidinal (Compound 5)

lucidenal;

Brown viscous liquid;

-25.3°(c 0.170, CHCl₃),

-26.7°(c 0.33, MeOH);

_{-25.3 ° (c 0.170, CHCl 3} ),

-26.7 [deg.] ( C 0.33, MeOH);

_{UV (CHCl 3) λ max ㎚} (log ε): 244 (2.75), 281 (2.74);

IR ? _Max cm ^-1 : 3425, 2926, 1728, 1680, 1642, 1514, 1268, 1228;

¹ H NMR (Methanol- d ₄ , 600 MHz) ?: 4.17 (1H, d, J = 11.8 Hz, H-1), 3.89 ), 6.16 (1H, s, H-4b), 9.39 (1H, s, H-5), 4.12 (1H, dd, J = 2.6, 9.8 Hz, H-6a), 3.90 (1H, dd, J = (1H, d, J = 8.1 Hz, H), 6.98 (1H, d, J = -3 '), 6.86 (1H, d, J = 1.6, 8.1 Hz, H-2'), 6.79 (1H, d, J = 1.3 Hz, H-2 ''), 6.62 (1H, d, J = 8.2 Hz, H-5 '' ), 6.65 (1H, dd, J = 1.3, 8.2 Hz, H-6 ''), 3.87 (3H, s, OCH 3), 3.80 (3H, s, OCH 3);

^{_{13 C NMR (Methanol- d 4,}} 150 MHz) δ: 52.8 (C-1), 41.9 (C-2), 151.4 (C-3), 138.7 (C-4), 196.3 (C-5), 67.1 (C-6), 172.7 (C-7), 20.8 (C-8), 136.4 (C-1 ') 112.8 , 116.1 (C-5 ') , 121.7 (C-6'), 56.6 (OCH 3), 136.3 (C-1 ''), 112.5 (C-2 ''), 148.9 (C-3 ''), 146.4 (C-4 '') , 116.1 (C-5 ''), 121.5 (C-6 ''), 56.5 (OCH 3);

HR-EI-MS m / z [M] + 400.1515 (calcd. For C 22 H 24 O 7).

Examples 2-6. 9,9'-bisacetyl-neo-olvyl (Compound 6)

9,9'-bisacetyl- neo- nonyl;

Pale powder;

UV (MeOH) [ lambda] _max nm (log [ epsilon] ): 225, 275;

IR ? _Max cm ^-1 : 3300, 2940, 2870, 1705, 1660, 1575, 1490, 1240, 1185, 1070;

^{_{1 H NMR (Methanol- d 4,}} 400 MHz) δ: 7.01 (2H, d, J = 1.8 Hz, H-2 ', 2''), 6.81 (2H, d, J = 8.1 Hz, H-5' , 5 ''), 6.89 (2H, dd, J = 1.8, 8.1 Hz, H-6 ', 6''), 4.95 (2H, d, J = 8.2 Hz, H- , m, H-3, 4 ), 4.27 (4H, d, J = 3.2 Hz, H-6, 7), 3.89 (6H, s, 2 'OCH 3, OCH 3), 1.91 (3H, s, COCH ₃ );

^{_{13 C NMR (Methanol- d 4,}} 100 MHz) δ: 134.3 (C-1, 1 '), 111.4 (C-2, 2'), 149.3 (C-3, 3 '), 147.7 (C-4, 4 '), 116.3 (C-5, 5'), 120.7 (C-6, 6 '), 85.1 _{7) 56.6 (OCH 3),} 172.7 (COCH 3), 20.8 (OCOCH 3);

EI-MS m / z [M] ⁺ 460 (calcd. For C ₂₄ H ₂₈ O ₉ ).

Examples 2-7. (2 S , 3 S ,4 S , 5 S ) -3,4-furandimethanol, tetrahydro-2,5-bis (4-hydroxy-3-methoxyphenyl) -3,4- diacetate (Compound 7)

(2 S, 3 S, 4 S, 5 S) -3,4-furandimethanol, tetrahydro-2,5-bis (4-hydroxy-3-methoxyphenyl) -3,4-diacetate;

Pale powder;

^{_{1 H NMR (Methanol- d 4,}} 400 MHz) δ: 7.11 (1H, d, J = 1.5 Hz, H-2 '), 7.03 (1H, d, J = 1.3 Hz, H-2''), 6.99 (1H, dd, J = 1.5 , 8.1 Hz, H-5 '), 6.90 (1H, d, J = 1.3, 8.1 Hz, H-5''), 6.86 (1H, d, J = 8.1 Hz, H -6 '), 6.83 (1H, d, J = 8.1 Hz, H-6''), 5.15 (1H, d, J = 7.6 Hz, H-2), 4.66 (1H, d, J = 8.5 Hz, H-5), 2.74 (1H , m, H-3), 2.42 (1H, m, H-4), 4.30 (2H, m, H-6, 7), 3.93 (3H, s, OCH 3), _{3.89 (3H, s, OCH 3} ), 1.97 (3H, s, OCOCH 3), 1.89 (3H, s, OCOCH 3);

^{_{13 C NMR (Methanol- d 4,}} 100 MHz) δ: 133.3 (C-1), 131.0 (C-1 '), 111.8 (C-2), 111.4 (C-2'), 149.3 (C-3) , 149.0 (C-3 '), 147.8 (C-4), 147.3 (C-4'), 116.4 6 '), 84.8 (C-7), 82.6 (C-7'), 46.7 (C-8), 51.3 _{OCH 3), 172.8 (OCOCH 3} ), 172.7 (OCOCH 3), 20.8 (OCOCH 3), 20.7 (OCOCH 3).

Examples 2-8. Echinoclorin A (Compound 8)

Echinochlorin;

White amorphous powder;

+20.2°(c 0.005, CHCl₃);

+ 20.2 ° (c 0.005, CHCl 3);

_{UV (CHCl 3) λ max ㎚} (log ε): 244 (4.55), 292 (4.52);

IR (KBr) ? _Max cm ^-1 : 3399, 2916, 2850, 1715, 1630, 1596, 1516, 1466, 1267, 1170;

&Lt; ¹ &gt; H NMR and &lt; ¹³ &gt; C NMR data are shown in Table 3 below;

HR-FAB-MS m / z 503.2595 [M + Na] ⁺ (calcd for C ₂₆ H ₄₀ O ₈ Na ⁺ ).

Examples 2-9. Echinoclorin B (Compound 9)

Echinochlorin B;

Pale amorphous powder;

UV (MeOH) ? _Max nm (log ? ): 203 (4.39), 278 (4.64);

IR (KBr) ? _Max cm ^-1 : 3441, 2928, 1676, 1666, 1583, 1407, 1240, 1076, 996, 990;

&Lt; ¹ &gt; H NMR and &lt; ¹³ &gt; C NMR data are shown in Table 3 below;

HR-FAB-MS m / z 309.2063 [M + H] ⁺ (calcd for C ₂₆ H ₄₁ O ₈ ⁺ ).

Examples 2-10. 2 ', 3'-dihydroxypropynonadecanoate (Compound 10)

2 ', 3'-dihydroxypropyl nonadecanoate;

White powder;

^{1 H NMR (Chloroform- d, 600} MHz) δ: 4.13 (2H, m, H-1 '), 3.89 (1H, m, H-2'), 3.65 (1H, dd, J = 3.7, 11.5 Hz, H-3'a), 3.56 (1H, dd, J = 5.9,11.5 Hz, H-3'b), 1.59 (2H, dd, J = 6.7, 7.0 Hz, H-2), 1.22-1.27 , m, H-2 ~ 18), 0.85 (3H, t, J = 6.8 Hz, H-19);

^{13 C NMR (Chloroform- d, 150} MHz) δ: 65.3 (C-1 '), 70.5 (C-2'), 63.6 (C-3 '), 174.6 (C-1), 22.9 (C-2) , 25.1 (C-3), 32.1 (C-4), 30.0 (C-5), 29.9 (C-6), 29.8 29.3 (C-10), 29.3 (C-11), 29.5 (C-12), 29.5 (C-17), 27.4 (C-18), 14.3 (C-19);

GC-MS m / z 354 [MH ₂ O] &lt; ^{+ &} gt ^; .

Examples 2-11. (8 E , 11 E ) -2 ', 3'-dihydroxypropyloctadeca-8,11-dienoate (Compound 11)

(8 E, 11 E) -2 ', 3'-dihydroxypropyloctadeca-8,11-dienoate;

Pale powder;

_{^{1 H NMR (Methanol- d 4,}} 600MHz) δ: 3.51 (2H, t, J = 4.5 Hz, H-1 '), 3.77 (1H, m, H-2'), 4.10 (1H, dd, J = 4.2, 11.4 Hz, H-3'a), 4.02 (1H, dd, J = 6.2,11.3 Hz, H-3'b), 2.31 (2H, t, J = 7.4 Hz, (2H, m, H-3), 1.31 (14H, m, H-4-6, 14-17), 2.02 , 11, 12), 2.73 (2H, t, J = 6.7 Hz, H-10), 0.87 (3H, t, J = 6.42 Hz, H-18);

¹³ C NMR (Methanol- d ₄ , 150 MHz) ?: 66.6 (C-1 '), 71.3 (C-2'), 64.2 , 26.1 (C-3), 32.8 (C-4), 30.8 (C-5), 30.6 (C-6), 28.3 (C-15), 30.3 (C-16), 23.7 (C-10), 129.2 (C-11), 131.0 (C-17), 14.6 (C-18);

GC-MS m / z 336 [MH ₂ O] &lt; ^{+ &} gt ^; .

Position Compound 1 (CD ₃ OD) 隆 _H ( J in Hz) δ _C One 132.6 2 6.87 (1H, dd, 1.9, 8.3) 121.1 3 6.91 (1H, d, 8.3) 118.7 4 149.6 5 149 6 7.01 (1H, d, 1.9) 111.7 7 6.55 (1H, d, 15.9) 135 8 6.18 (1H, dt, 6.5, 15.9) 123.2 9 4.64 (2H, dd, 1.1, 6.5) 64.9 One' 133.4 2' 6.82 (1H, dd, 1.8, 8.3) 121 3 ' 6.72 (1H, d, 8.3) 116 4' 147.5 5 ' 152 6 ' 6.98 (1H, d, 1.8) 111.9 7 ' 4.81 (1H, d, 5.8) 74.5 8' 4.52 (1 H, m) 83.4 9'a 4.18 (1H, dd, 3.7, 11.9) 66.4 9'b 3.97 (1H, dd, 6.2, 11.9) OCH ₃ 3.78 (3 H, s) 56.5 OCH ₃ 3.88 (3 H, s) 56.7 OCO CH ₃ 1.87 (3 H, s) 21 OCO CH ₃ 1.88 (3 H, s) 20.7 O C OCH ₃ 172.6 O C OCH ₃ 172.9 ¹ H (600 MHz) and ¹³ C (150 MHz) NMR

Position 1S ^b 1R ^b δ _H δ _H 7 ' 6.35 (d, J = 6.2)
or 6.27 (d, J = 6.2) 6.27 (d, J = 6.2)
or 6.35 (d, J = 6.2) 8' 4.68 (m) or 4.64 (m) 4.64 (m) or 4.68 (m) 9'a 4.20 (dd, J = 4.5, 12.0)
or 4.13 (dd, J = 4.5, 12.0) 4.13 (dd, J = 4.5, 12.0)
or 4.20 (dd, J = 4.5, 12.0) 9'b 3.90 (dd, J = 5.2, 12.0)
or 3.85 (dd, J = 5.2, 12.0) 3.85 (dd, J = 5.2, 12.0)
or 3.90 (dd, J = 5.2, 12.0) ? in ppm, J in Hz; ^b in chloroform- d

Position Compound 8 (CDCl ₃₎ Compound 9 (CD ₃ OD) 隆 _H ( J in Hz) δ _C 隆 _H ( J in Hz) δ _C One 4.32-4.40 (2H, overlapped) 64.8 177.8 2 1.83 (2H, m) 29.8 2.20 (2H, t, 7.4) 35.0 3 1.90 (2H, m) 29.7 1.50-1.55 (2H, m) 26.1 4 1.46-1.60 (2H, m) 26.2 1.22-1.29 (2H, m) 30.1 5 1.46-1.60 (2H, m) 28.9 1.22-1.29 (2H, m) 30.0 6 1.46-1.60 (2H, m) 29.3 1.50-1.55 (2H, m) 25.0 7 1.46-1.60 (2H, m) 29.4 2.56-2.61 (2H, overlapped) 41.8 8 1.46-1.60 (2H, m) 29.6 202.8 9 1.46-1.60 (2H, m) 29.7 6.48-6.52 (1 H, m) 137.5 10 1.46-1.60 (2H, m) 29.8 7.20-7.24 (1 H, m) 141.0 11 1.83 (2H, m) 25.1 7.20-7.24 (1 H, m) 141.0 12 2.55 (2H, t, 6.1) 34.3 6.48-6.52 (1 H, m) 137.5 13 174.5 202.7 14 2.56-2.61 (2H, m) 41.8 15 1.50-1.55 (2H, m) 25.1 16 1.22-1.29 (2H, m) 32.7 17 1.22-1.29 (2H, m) 23.7 18 0.83 (3H, t, 6.9) 14.4 One' 148.2 2' 7.12 (1H, d, 8.0) 115.0 3 ' 7.27 (1H, d, 8.0) 123.2 4' 127.2 5 ' 7.24 (1H, overlapped) 109.6 6 ' 147.0 7 ' 7.81 (1H, d, 15.8) 144.9 8' 6.50 (1H, d, 15.8) 115.8 9 ' 167.7 OCH ₃ 4.13 (3H, overlapped) 56.1 One'' 4.32-4.40 (2H, overlapped) 65.3 2'' 4.13 (1H, overlapped) 70.4 3''a 3.80 (1H, dd, 4.9, 9.8) 63.6 3''b 3.90 (1H, br, d, 9.8)

< Example  3. Cell culture>

The culture conditions of the cells used for measuring the anti-inflammatory activity against the compounds isolated from edible fatigue of the present invention are as follows.

Mouse macrophages RAW 264.7 cells were cultured in DMEM (Dulbecco's Modified Essential Medium) medium containing 10% FBS (fetal bovine serum), penicillin (100 U / ml) / streptomycin _{2. &} Lt; / RTI &gt; The cultured cells were inoculated in 6-well tissue culture plates at a concentration of 2.5 × 10 ⁶ cells / well, and the compounds 1 to 11 of the present invention were added at a concentration of 12.5 to 50.0 μg / ml , Preincubated for 60 min.

&Lt; Example 4: Measurement of cell viability &

(3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) assay was conducted to measure the cell viability of the compounds 1 to 11 of the present invention.

First, compounds 1 to 11 of the present invention were treated at a concentration of 50 / / ml in RAW 264.7 cells (5 × 10 ⁴ cells / ml) cultured under the conditions of Example 3, and treated with 0.1 μg / ml of LPS Lt; / RTI &gt; When the control (untreated group) was treated, the cells were stimulated with 0.1 μg / ml of LPS for 24 hours or cultured for 24 hours without LPS treatment. After 24 hours, 50 쨉 l of 2 mg / ml MTT reagent was added to the culture, followed by further reaction at 37 캜 for 2 hours. The formazan crystal produced by the above procedure was dissolved in DMSO and the absorbance at 570 nm was measured and shown in FIG.

Referring to FIG. 2, in the case of the compounds 1 to 11 of the present invention and the control group, the cell survival rate in all treatment groups was 95% or more, and the compounds 1 to 11 of the present invention did not show cytotoxicity to macrophages I could confirm.

&Lt; Example 5: Analysis of NO production inhibitory activity &

(Nitrite) was measured using the supernatant of the cultured RAW 264.7 cells, and the NO production amount was analyzed by referring to the preceding document [Migliorini, P. et al., 1991] .

First, RAW 264.7 cells (5 x 10 ⁴ cells / ml) cultured under the conditions of Example 3 were treated with the compounds 1 to 11 of the present invention (12.5, 25 and 50.0 μg / ml of each concentration) or a positive control Treated with dexamethasone (0.2, 0.4 and 0.8 μg / ml each concentration), and then stimulated with 0.1 μg / ml of LPS for 24 hours. When the negative control (untreated group) was treated , Stimulated with 0.1 μg / ml of LPS for 24 hours as described above, or cultured for 24 hours without LPS treatment. Then, the cell supernatant was mixed with an equal amount of a grease reagent, and the absorbance at 570 nm was measured. From this, the NO production inhibitory activity was analyzed and shown in FIG. 3 and Table 4.

Condition IC ₅₀ (uM) ^a Compound 1 101.75 ± 7.60 Compound 2 51.91 + - 4.74 Compound 3 36.03 + - 4.75 Compound 5 4.80 + - 0.50 Compound 6 27.16 + - 4.35 Compound 9 20.77 ± 1.62 Compound 11 60.12 + 2.83 The positive control (dexamethasone) 1.02 0.08 * ^a : The inhibitory effects are expressed as concentration (μM) giving 50% inhibition (IC ₅₀ ) relative to the control.

Referring to Table 4 and FIG. 3, when the compound of the present invention is treated with LPS-treated macrophages, the amount of NO produced decreases in a concentration-dependent manner, indicating that the compound of the present invention exhibits NO production inhibitory activity . Thus, the compounds of the present invention can be effectively used as a composition for the prophylaxis or treatment of inflammatory diseases, and the NO production inhibitory activity against Compounds 4, 7, 8 and 10 is also shown in Table 4 and FIG. 3 I could confirm.

&Lt; Example 6: Immunoblot analysis >

To examine the effect of the compounds of the present invention on iNOS and COX-2, proteins involved in inflammation, the protein was extracted from macrophages and expressed and analyzed.

(50 mM Tris-HCl, pH 7.5, 1% Nonidet P-40, 1.0 mM EDTA, 1.0 mM phenylmethylsulfonyl fluoride, 1.0 μg / mL leupeptin, 1.0 mM sodium vanadate, 150 mM NaCl) was added to extract the protein. 50 μg of the extracted protein was subjected to SDS-PAGE and transferred to PVDF (polyvinylidene difluoride, Millipore, Bedford, MA, USA). The membranes were blocked with PBST (phosphate buffered saline-Tween buffer) containing 5% skim milk at 25 ° C for 30 minutes and then incubated with primary antibodies (iNOS and COX-2, Santa Cruz, CA , USA) was added, followed by further reaction. Thereafter, the reaction was carried out with a secondary antibody containing HRP (horseradish peroxidase), followed by analysis of the protein-transferred membrane by adding ECL reagent (Amersham Pharmacia Biotec, UK).

4, when the compound of the present invention is treated with LPS-treated macrophages, the effect of inhibiting the expression of inflammatory-related proteins iNOS and COX-2 in a concentration-dependent manner is excellent, May be useful as a composition.

&Lt; Example 7: Toxicity test &

Example 7-1. Acute toxicity

This experiment was carried out to investigate the toxicity of the compound 9 (echinoclorin B) of the present invention to an animal body acutely (within 24 hours) when an excessive amount of the compound 9 of the present invention was ingested in a short period of time and to determine the mortality. Twenty ICR mice were prepared, and 10 mice were assigned to each group. In the control group, 30% PEG-400 alone was administered, and in the experimental group, Compound 9 (echinoclorin B) of the present invention was orally administered at a concentration of 1.0 g / kg each. After 24 hours of administration, the respective mortality rates were examined. As a result, both the control group and the test group to which Compound 9 (echinoclorin B) was administered survived.

Example 7-2. Organ organs toxicity test in experimental group and control group

The long-term toxicity test was carried out on C57BL / 6J mice in order to investigate the effects of each compound (9) (echinoclorin B) of the present invention at a concentration of 1.0 g / After 8 weeks from the control animals, blood samples were collected and the blood concentrations of GPT (glutamate-pyruvate transferase) and BUN (blood urea nitrogen) were measured using Select E (vital scientific NV, Netherland). As a result, GPT, which is known to be related to hepatotoxicity, and BUN, which is known to be related to renal toxicity, showed no significant difference compared to the control group. In addition, liver and kidney were cut from each animal and histological observation was performed with an optical microscope through a conventional tissue section production process, but no abnormal abnormalities were observed.

&Lt; Formulation Example 1 >

Formulation Example 1-1. Manufacture of tablets

200 g of the compound 9 of the present invention (echinoclorin B) was mixed with 175.9 g of lactose, 180 g of potato starch and 32 g of colloidal silicic acid. To this mixture was added a 10% gelatin solution, which was pulverized and passed through a 14-mesh sieve. This was dried, and a mixture obtained by adding 160 g of potato starch, 50 g of talc and 5 g of magnesium stearate was made into tablets.

Formulation Example 1-2. Injection preparation

1 g of the compound 9 (echinoclorin B) of the present invention, 0.6 g of sodium chloride and 0.1 g of ascorbic acid were dissolved in distilled water to make 100 ml. This solution was placed in a bottle and sterilized by heating at 20 DEG C for 30 minutes.

<Formulation Example 2: Food Preparation>

Formulation Example 2-1. Manufacture of cooking seasonings

Compound 9 (echinoclorin B) of the present invention was added to the cooking sauce in an amount of 1 wt% to prepare a cooking sauce for health promotion.

Formulation Example 2-2. Manufacture of flour food products

A food for health promotion was prepared by adding the compound 9 (echinoclorin B) of the present invention to wheat flour at 0.1 wt% and preparing bread, cake, cookies, crackers and noodles using this mixture.

Preparation Example 2-3. Manufacture of soups and gravies

Health enhancing soup and juice were prepared by adding Compound 9 (echinoclorin B) of the present invention to soup and juice at 0.1 wt%.

Formulation Example 2-4. Manufacture of dairy products

Compound 9 (echinoclorin B) of the present invention was added to milk in an amount of 0.1 wt%, and various dairy products such as butter and ice cream were prepared using the milk.

Formulation Example 2-5. Vegetable juice manufacturing

0.5 g of the compound 9 of the present invention (echinoclorin B) was added to 1,000 ml of tomato juice or carrot juice to prepare vegetable juice for health promotion.

Formulation example  2-6. Manufacture of fruit juice

0.1 g of the compound 9 of the present invention (echinoclorin B) was added to 1,000 ml of apple juice or grape juice to prepare fruit juice for health promotion.

Claims (7)

A composition for preventing or treating an inflammatory disease, which comprises, as an active ingredient, echinoclorin B (Compound 9) of the following formula 1 isolated from edible blood ( Echinochloa utilis ).
[Chemical Formula 1]

The method according to claim 1,
Said inflammatory disease is selected from the group consisting of allergic diseases, inflammatory bowel disease, atherosclerosis, inflammatory collagen vascular disease, glomerulonephritis, inflammatory skin disease, sarcoidosis, retinitis, gastritis, hepatitis, enteritis, arthritis, tonsillitis, sore throat, bronchitis, pneumonia, pancreatitis, Sepsis and nephritis. &Lt; RTI ID = 0.0 &gt; 21. &lt; / RTI &gt; A health functional food for preventing or ameliorating an inflammatory disease, which comprises, as an active ingredient, echinoclorine B (Compound 9) of the following formula 1 isolated from edible blood ( Echinochloa utilis ).
[Chemical Formula 1]

The method of claim 3,
Said inflammatory disease is selected from the group consisting of allergic diseases, inflammatory bowel disease, atherosclerosis, inflammatory collagen vascular disease, glomerulonephritis, inflammatory skin disease, sarcoidosis, retinitis, gastritis, hepatitis, enteritis, arthritis, tonsillitis, sore throat, bronchitis, pneumonia, pancreatitis, Septicemia, and nephritis. &Lt; RTI ID = 0.0 &gt; 18. &lt; / RTI &gt; delete A novel compound echinoclorin B (compound 9) of the formula 1
[Chemical Formula 1]

Extracting edible blood ( Echinochloa utilis ) with water, C1 to C4 lower alcohol or a mixed solvent thereof to prepare an edible blood extract; Sequentially fractionating the edible extract using n-hexane, methylene chloride, ethyl acetate and n-butanol; And separating the fractions from each other by chromatography to obtain an echinoclorin B (compound 9) of the following formula (1).
[Chemical Formula 1]

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