KR20140016217A - Composition Comprising and healthy food Luteolin 5―O―β―D―glucopyranoside or the Extract of Cirsium maackii for Preventing or Treating Inflammatory Disease - Google Patents

Abstract

The present invention relates to a pharmaceutical composition and a health functional food containing raw extract and non polar solvent-soluble extract of Cirsium maackii, or a compound of luteolin 5-O-β-D-glucopyranoside for preventing or treating inflammatory diseases. An active ingredient has an anti-inflammatory activity in lipopolysaccharides-induced nitrogen oxide generation of RAW 264.7 macrophage and right leg edema in carrageenan-induced mice. Thus, the Cirsium maackii extract has a strong preventive activity against the inflammatory diseases and secures the safety as a natural medicinal herb, thereby being able to be useful as the pharmaceutical composition and health functional food for preventing or treating the inflammatory diseases.

Description

Compositions Comprising and Healthy Foods for Preventing or Treating Inflammatory Diseases Containing Thistle Extract or Compounds of Luteolin 5-O-β-D-Glucopyranoside Isolated from Luteolin 5-O-β ―D―glucopyranoside or the Extract of Cirsium maackii for Preventing or Treating Inflammatory Disease}

The invention in inflammatory disorders, containing a compound of thistle crude extract, non-polar solvent soluble extract or from a separate base Theo Lin 5- O -β-D- glucopyranoside (luteolin 5- O -β-D- glucopyranoside) It relates to a pharmaceutical composition for the prevention or treatment and a dietary supplement.

Inflammation is one of the defense responses of biological tissues to external stimuli and is a complex lesion that involves tissue degeneration, circulatory disorders, and tissue proliferation (see Non-Patent Document 1). These inflammatory processes are mainly mediated by external stimulants such as lipopolysaccharides (LPS) or internal stimulants such as arachidonic acid metabolites, macrophages, granulocytes, and fibroblasts. Influx and accumulation of inflammatory cells, such as fibroblasts, into the site of inflammation (see Non-Patent Document 2). In particular, activated macrophages in the inflammatory site play a large role in mediating inflammation by producing large amounts of prostaglandins (PGs) and nitric oxide (NO), which are not only cytokines but also arachidonic acid metabolites. do.

Inflammation causes various biochemical phenomena, but especially nitric oxide synthase (NOS), an enzyme that produces NO, and cyclooxygenase, an enzyme that mediates the biosynthesis of various PGs. COX) is known as an important mediator of inflammatory responses (see Non-Patent Documents 3, 4, 5).

NO is produced by constitutive nitric oxide synthase (cNOS) and inducible nitric oxide synthase (iNOS) from L-arginine, which is a constant expression of low levels of NO that mediates physiological functions. INOS is rapidly induced by stimuli such as LPS and cytokines to generate excess NO (see Non-Patent Document 6). Therefore, NO production in the inflammatory response is mostly due to iNOS (see Non-Patent Document 7). PGs are produced from arachidonic acid by COX-1 (constitutive cyclooxygenase) and inducible cyclooxygenase (cyclooxygenase-2, COX-2).

COX-1, like cNOS, is constantly expressed and regulates physiological functions, whereas COX-2 is an inflammatory stimulant that causes macrophages, granulocytes, fibroblasts and It is derived from the same inflammatory cells (see Non-Patent Document 8), and also PGs in the inflammatory response are mostly produced by COX-2 (see Non-Patent Document 9).

Macrophages play an important role in the first line of host defense against external pathogens, as well as neutrophills and natural killer cells. When infections and inflammation are produced, L-arginine is produced as NO by iNOS, and activated macrophages secrete NO and pro-inflammatory cytokines to other immune cells. And tissue (see Non-Patent Document 10). However, excessive inflammatory reactions cause inflammatory diseases such as septic shock or rheumatoid arthritis, and it has been reported that excessive production of NO and PGs plays an important role in this process (Non-Patent Document 11). Reference). Accordingly, a substance capable of inhibiting excessive production of NO and PGs and expression of enzymes involved in their production has attracted attention as an agent for preventing and treating inflammatory diseases. In particular, the components separated and purified from food are relatively less toxic and safer for long-term intake than pharmaceuticals, and thus are receiving attention as treatments and supplements for various inflammatory diseases. Superoxide (Superoxide, O ₂ ^-), hydrogen peroxide _{(hydrogen peroxide, H 2 O 2} ), hydroxyl radicals (hydroxyl radical, OH), singlet oxygen _{^{(singlet oxygen, 1 O 2)}} , an alkoxyl radical ( reactive oxygen species (ROS) and reactive nitrogen species (RNS), such as alkoxyl radicals (RO ·), peroxyl radicals (ROO), and peroxynitrite (ONOO ⁻ ). ) Is produced from inflammatory cells such as macrophages. These ROS and RNSs act as powerful oxidative and nitrifying elements that damage our body's components, such as lipids, proteins, nucleic acids, and DNA, resulting in chronic diseases associated with inflammation and aging (non-patents). See Documents 12, 13).

In addition, ROS and RNS influence the conversion of arachidonic acid to inflammatory mediators by cyclooxygenase and lipooxygenase, and during the inflammatory process by macrophages. Free radicals are produced. Therefore, it can be expected that antioxidants capable of eliminating free radicals can prevent and treat diseases caused by inflammation and oxidative stress (see Non-Patent Document 14).

Thistle ( Cirsium maackii ) is a perennial herb of the Asteraceae, and grows in the mountain families of Korea. As a medicinal herb, it is called the grand system and it is dried thistle outpost, and it has the effect of bleeding haemostasis, blood detoxification, and anti-inflammatory effect (see Non-Patent Documents 15 and 16). The roots are harvested in autumn, and the leaves and stems are harvested at the time of flowering, dried in the sun and chopped before use. Thistle is about 1 m high, with white hairs, spider-like hairs, and branched branches. Root leaves remain until flowering, larger than neuritis, oval or lanceolate oval, 15-30 cm long, 6-15 cm wide, narrow at the bottom, split into 6-7 pairs of idols, hairy on both sides, edge The spines are lanceolate, with lanceolate serrates on the lanceolate, lanceolate oval, rounded to the main stem, and the edges split into idols are split again. Flowers bloom in June-August, flowers are purple or red, 3 ~ 5 cm in diameter, hang at the ends of branches and stems, round guns are 18-20 mm long, 25-35 mm in diameter, and fragments are arranged in 7-8 rows. It is slightly longer from the outside to the inside, and has a pointed linear shape. The corolla is 19 ~ 24 mm long. Fruits mature in August, achenes are 3.5-4 mm in length, tubules 16-19 mm in length. Distributed throughout the country and grows in mountains and fields. Thistle (larvae) has been reported to have a hemostatic action, the main component that exhibits a hemostatic action is known as pectolinarin (pectolinarin), the dose-dependent reduction of the bleeding time is observed in a mouse experiment (see Non-Patent Document 17). Large scales have activity on the cardiovascular system and are reported to show a drop in blood pressure at the extract level (see Non-Patent Document 18). On the other hand, an increase in blood pressure is observed during intramuscular injection or intravenous injection, but no effect of blood pressure increase is observed during oral administration (see Non-Patent Document 19). Vasodilation was observed via the histamine H1 receptor when treated with extract water in rat thoracic aorta (see Non-Patent Document 20). Pectolinarin, 5,7-dihydroxy-6,4-dimethoxyflavone, among the flavone components in the larvae, S180, H22 tumors When the mice were transplanted into mice, both tumors were inhibited in proliferation, lymphocyte proliferation was observed, and NK cell activity was increased. ). It is reported that there is an antibacterial activity in the large system, and that the isolated polyacetylene promotes the differentiation of neurites in PC12h cells and Neuro2a cells, and that memory impairment induced by scopolamine in mice is significantly improved. (See Non-Patent Document 22). Large water extracts are known to have an effect similar to that of the female hormone estrogen, activating estrogen receptors and increasing the expression of progestrone and pS2 genes (see Non-Patent Document 23). It has the efficacy of antipyretic, hemostasis, small swelling, etc.It is used for the treatment of colds, whooping cough, high blood pressure, enteritis, nephritis, hemostasis, hematuria, bloody stool, and postpartum bleeding, hyperhidrosis and boils (see Non-Patent Documents 24 and 25). ). In particular, thistle is a herb that is similar in activity and homology to Silybum marianum , known as milk thistle, and is reported to have an excellent effect on hepatoprotective effects, and is currently used as a hepatoprotectant. (See Non-Patent Documents 26 and 27). In addition, antibacterial (see Non-Patent Document 28), antidiabetic (see Non-Patent Document 29), antioxidant (see Non-Patent Document 30), anti-inflammatory (see Non-Patent Document 31), vasorelaxation (see Non-Patent Document 32) , Anticancer activity (see Non-Patent Document 33), and anti-diabetic complications have been reported.

The components that have been identified so far include essential oils and triterpene compounds.As essential oils, aflotaxene, dihydroaplotaxene, tetrahydroaplotaxene and hexahydroapple Rotaxene (hexahydroaplotaxene), cyperene (cyperene), caryophyllene (caryophyllene), thujopsene (alpha-himachalene), etc. are separated and alpha-amirin as a triterpene compound (α-amyrin), beta-amyrin, and the like are separated (see Non-Patent Document 34), and recently, polyacetylene compounds have been separated (see Non-Patent Document 22). Compounds isolated or identified from thistles are flavonoids, phenolic acids, lignans, polyacetylenes, acetylenes, sterols, triterpenes, sesquis There are terpene lactones (sesquiterpene lactones) and alkaloids (see Non-Patent Documents 35, 36, 37, 38) and these are believed to be involved in various physiological activities. In particular, it was reported that native thistle contains a large amount of various flavonoids related to antioxidant (see Non-Patent Document 30).

Thus, it has been reported that flavonoids, tannins, coumarins, essential oils and the like have an inhibitory effect on activity in cells, cows, rats or humans. However, the preventive and protective effects of thistle extract against inflammatory diseases have not been reported until now, and the anti-inflammatory effects of pectolinarin and pectolinarigenin isolated from Cirsium chanroenicum by Lim et al. Only non-patent document 39) is known (see non-patent document 31), and luteolin and luteolin 7-glucoside (see non-patent document 39) are only known. Therefore, the present inventors have proposed a novel extract and luteolin 5-glucopyranoside (luteolin) for the prevention and treatment of inflammatory diseases with nitric oxide production of lipid polysaccharide-induced RAW 264.7 macrophages in milk thistle and right foot inflammation in carrageenan-induced mice. 5- O- β-D-glucopyranoside) compounds were isolated and identified, and their anti-inflammatory effects were completed to complete the present invention. In addition, since the anti-inflammatory activity of this compound has not been determined, no studies have been conducted on LPS-induced NO production, iNOS and COX-2 expression, and t- BHP-induced ROS production inhibition in RAW 264.7 cells. Their anti-inflammatory activity was studied.

[Non-Patent Document 1] Tizard, I. R., Immunology: An introduction inflammation. 2nd ed. Saunders College Pub. Co. New York, 423-444, 1986. Non Patent Literature 2: Trowbridge, H. O. and Emling, R. C., Inflammation: a review of the process, 5th ed. Quintessence Pub. Co. Chicago, 3-9, 1997. Non-Patent Document 3: Lawrence, T., Wiilloughby, D. A. and Gilroy, D. W., Anti-inflammatory lipid mediators and insights into the resolution of inflammation, Nat. Rev. Immunol., 2, 787-795, 2002. Non Patent Literature 4: Higuchi, M., Hisgahi, N., Taki, H., Osawa, T., Cytolytic mechanisms of activated macrophages. Tumor necrosis factor and l-arginine-dependent mechanisms act synergistically as the major cytolytic mechanisms of activated macrophages, J. Immunol., 144, 1425-1143, 1990. Non Patent Literature 5: Willeaume, V., Kruys, V., Mijatovic, T., Huez, G., Tumor necrosis factor-alpha production induced by viruses and by lipopolysaccharide in macrophages: similarities and differences. J. Inflamm. 1996; 46 (1): 1-12. Non-Patent Document 6: Liang, YC, Huang, YT, Tsai, SH, Lin-Shiau, SY, Chen, CF, Lin, JK, Suppression of inducible cyclooxygenase and inducible nitric oxide synthase by apigenin and related flavonoids in mouse macrophages, 20, 1945-1952, 1999. Non Patent Literature 7: Huang, YC, Guh, JH, Cheng, ZJ, Chang, YL, Hwang, TL, Liao, CH, Tzeng, CC, Teng, CM, Inhibition of the expression of inducible nitric oxide synthase and cyclooxygenase-2 in macrophages by 7HQ derivatives: involvement of Ikappa B-alpha stabilization ,, 418, 133--139, 2001. [Non-Patent Document 8] Herschman, H. R., Prostaglandin synthase 2, Biochim. Biophys. Acta., 1299, 125-140, 1996. Non Patent Literature 9: Reddy, S. T. and Herschman, H. R., Ligand-induced prostaglandin synthesis requires expression of the TIS10 / PGS-2 prostaglandin synthase gene in murine fibroblasts and macrophages, 269, 15473-15480, 1994. Non-Patent Document 10: Nathan, C. and Xie, Q. W., Nitric oxide synthases: roles, tolls, and controls, Cell, 78, 915, 1994. Non Patent Literature 11: Higgs, G. A., Moncada, S. and Vane, J. R., Eicosanoids in inflammation,, 16,287-99, 1984. Non-Patent Document 12: (12) 8 Beckman, J. S., Beckman, T. W., Chen, J., Marshell, P. A., Freeman, B. A., Apparent hydroxyl radical production by peroxynitrite: implications for endothelial injury from nitric oxide and superoxide, Proc. Natl. Acad. Sci., 87, 1620-1624, 1990. [Non-Patent Document 13] Vallyathan, V., Shi, X., The role of oxygen free radicals in occupational and environmental lung diseases, Environ. Health Perspect., 105, 165-177, 1997. Non Patent Literature 14: Backhouse, N., Delporte, C., Givernau, M., Cassels, BK, Valenzuela, A., Speisky, H., Anti-inflammatory and antipyretic effects of boldine, Agents Actions., 42, 114 117, 1994. Non-Patent Document 15: Yuk Chang-soo, Kim Sung-man, Jung Jin-mo, Jung Myeong-sook, Kim Jung-hwa, Kim Seung-bae. Pharmacology, ingredients, clinical applications of Chinese medicine. Celebration Culture History. 1998. pp 594-595. [Non-Patent Document 16] Park, Jong-Hee, Lee, Jung-Kyu. Commercial medicinal plants illustrated. Shin Il Co. 2000. pp274-276. Non-Patent Document 17: Ishida H, Umino T, Tsuji K, Kosuge T. Studies on antihemorrhagic substances in herbs classified as hemostatics in Chinese medicine. VII. On the antihemorrhagic principle in Cirsium japonicum DC. Chem Pharm Bull (Tokyo). 1987; 35: 861-864. Non Patent Literature 18: Zhu YP. Chinese Materia Medica Chemistry, Pharmacology and Applications. Harwood Academic Publishers, The Netherlands, pp 405-406, 1998a. Non-Patent Document 19: Zhu YP. Chinese Materia Medica Chemistry, Pharmacology and Applications. Harwood Academic Publishers, The Netherlands, pp 403-405, 1998b. [Non-Patent Document 20] Kim EY, Jho HK, Kim DI, Rhyu MR. Cirsium japonicum elicits endothelium-dependent relaxation via histamine H (1) -receptor in rat thoracic aorta. J Ethnopharmacol. 2008; 116: 223-227. Non-Patent Document 21: Liu S, Zhang J, Li D, Liu W, Luo X, Zhang R, Li L, Zhao J. Anticancer activity and quantitative analysis of flavone of Cirsium japonicum DC. Nat Prod Res. 2007; 21: 915-922. Non-Patent Document 22: Yamazaki M, Hirakura K, Miyaichi Y, Imakura K, Kita M, Chiba K, Mohri T. Effect of polyacetylenes on the neurite outgrowth of neuronal culture cells and scopolamine-induced memory impairment in mice. Biol Pharm Bull. 2001; 24: 1434-1436. Non-Patent Document 23: Park MK, Rhyu MR, Yoon BK, Kwon H, Jang S, Lee YJ. Modulation of the genomic estrogen receptor pathway by water extracts of Cirsium japonicum. Arch Pharm Res. 2008; 31: 225-230. Non-Patent Document 24: Lee, Y. N. Flora of Korea. Kyo-Hak-Sa: Korea; 2002, pp 844. Non-Patent Document 25: Kim, J. G. Illustrated Natural Drugs Encyclopedia (color edition). Namsandang: Korea; 1997, Vol. One; pp 37. Non Patent Literature 26: Park, J. C .; Hur, J. M .; Park, J. G .; Kim, S. C .; Park, J. R .; Choi, S. H .; Choi, J. W. Effects of methanol extract of Cirsium japonicum var. ussuriense and its principle, hispidulin-7-O-neohesperidoside on hepatic alcohol-metabolizing enzymes and lipid peroxidation in ethanol-treated rats. Phytother. Res. 2004, 18, 19-24. Non Patent Literature 27: Ku, K. L .; Tsai, C. T .; Chang, W. M .; Shen, M. L .; Wu, C. T .; Liao, H. F. Hepatoprotective effect of Cirsium arisanense Kitamura in tacrine-treated hepatoma Hep 3B cells and C57BL mice. Am. J. Chin. Med. 2008, 36, 355-368. Non Patent Literature 28: Nazaruk, J .; Jakoniuk P. Flavonoid composition and antimicrobial activity of Cirsium rivulare (Jacq.) All. Flowers. J. Ethnopharmacol. 2005, 102, 208-212. Non Patent Literature 29: Perez G., R. M .; Ramirez, L. M. E .; Vargas, S. R. Effect of Cirsium pascuarense on blood glucose levels of normoglycaemic and alloxan-diabetic mice. Phytother. Res. 2001, 15, 552-554. Non Patent Literature 30: Jeong, da M .; Jung, H. A .; Choi, J. S. Comparative antioxidant activity and HPLC profiles of some selected Korean thistles. Arch. Pharm. Res. 2008, 31, 28-33. Non Patent Literature 31: Lim, H .; Son, K. H .; Chang, H. W .; Bae, K .; Kang, S. S .; Kim, H. P. Anti-inflammatory activity of pectolinarigenin and pectolinarin isolated from Cirsium chanroenicum. Biol. Pharm. Bull. 2008, 31, 2063-2067. Non Patent Literature 32: Kim, E. Y .; Jho, H. K .; Kim, D. I .; Rhyu, M. R. Cirsium japonicum elicits endothelium-dependent relaxation via histamine H (1) -receptor in rat thoracic aorta. J. Ethnopharmacol. 2008, 116, 223-227. Non Patent Literature 33: Liu, S .; Zhang, J .; Li, D .; Liu, W .; Luo, X .; Zhang, R .; Li, L .; Zhao, J. Anticancer activity and quantitative analysis of flavone of Cirsium japonicum DC. Nat. Prod. Res. 2007, 21, 915-922. Non-Patent Document 34: Sung CK. International Collation of Traditional and Folk Medicine. Kimura (Ed). vol. 1, pp 171-172, World Scietific Publishers, Singapore, 1996. [Non-Patent Document 35] Jordon-Thaden, I. E .; Louda, S. M. Chemistry of Cirsium and Carduus: a role in ecological risk assessment for biological control of weeds? Biochem. Syst. Ecol. 2001, 31, 1353-1396 [Non-Patent Document 36] Lee, B. L .; Kwan, J. H .; Zee, O. P .; Yoo, S. J. Flavonoids from Cirsium rhinoceros. Arch. Pharm. Res. 1994, 17, 273-277. Non Patent Literature 37: Yim, S. H .; Kim, H. J .; Lee, I. S. A polyacetylene and flavonoids from Cirsium rhinoceros. Arch. Pharm. Res. 2003, 26, 128-131. Non Patent Literature 38: Chung, A. K .; Kwon, H. C .; Choi, S. Z .; Min, Y. D .; Lee, S. O .; Lee, W. B .; Yang, M. C .; Lee, K. H .; Nam, J. H .; Kwak, J. H .; Lee, K. R. Norisoprenoids from Cirsium rhinoceros. Kor. J. Pharmacogn. 2002, 33, 81-84. [Non-Patent Document 39] Hu, C., Kitts, D. D., Luteolin and luteolin-7-O-glucoside from dandelion flower suppress iNOS and COX-2 in RAW 264.7 cells. Mol. Cell. Biochem., 265, 107-113 (2004).

In order to solve the above problems, the present invention contains a crude thistle extract or a compound of luteolin 5- O- β-D-glucopyranoside isolated therefrom as an active ingredient, and induces lipid polysaccharide. An object of the present invention is to provide a pharmaceutical composition and a dietary supplement for preventing or treating inflammatory diseases, which have nitric oxide production of RAW 264.7 macrophages and inhibitory activity on carrageenan induced mice.

The present invention to solve the above problems

(1) drying and grinding the thistle into powder;

(2) Methanol was added to the milk thistle powder prepared in step (1), and reflux-cooled extraction was performed three times at intervals of 12 hours, 6 hours, and 3 hours, respectively, at a temperature of 70 ° C., followed by filtration under reduced pressure, followed by 40 to 70 Concentrating under reduced pressure at ℃ to obtain a crude methanol extract; And

(3) dissolving the crude methanol extract obtained by the above step (2) in distilled water, dichloromethanol mixture was repeated three times, and then concentrated and dried under reduced pressure at 40 to 70 ℃ to obtain a dichloromethanol extract Manufactured, including;

Provided is a method for preparing an extract for preventing or treating inflammatory diseases, which comprises a 5-O-β-D-glucopyranoside compound represented by Formula 1 as an active ingredient. .

[Formula 1]

In the method for preparing an extract for preventing or treating an inflammatory disease of the present invention, the dichloromethanol extract obtained by the above step (3) is dissolved in distilled water, and ethyl acetate is mixed to repeat the above three times distribution, and 40 to 70 ° C. Concentrating and drying under reduced pressure at to obtain an ethyl acetate extract (4) characterized in that it further comprises a step.

In the method for producing an extract for preventing or treating inflammatory diseases of the present invention, the ethyl acetate extract obtained by the above step (4) is dissolved in distilled water, n -butanol is mixed, and the above-described distribution is repeated three times, 40 to 70 Concentrating and drying under reduced pressure at ℃ it is characterized in that it further comprises the step (5) to obtain n -butanol extract.

The present invention is prepared according to the production method of the present invention and is characterized by having nitric oxide production of lipid polysaccharide induced RAW 264.7 macrophages and inhibitory activity of right foot paw in carrageenan induced mice.

The compounds of the thistle crude extract, non-polar solvent soluble extract or from a separate base Theo Lin 5- O -β-D- glucopyranoside (luteolin 5- O -β-D- glucopyranoside) prepared by the production method of the present invention Has nitric oxide production in lipid polysaccharide-induced RAW 264.7 macrophages and inhibitory activity in the right foot of carrageenan-induced mice.

Therefore, the extract shows a strong inflammatory disease prevention activity and is secured as a natural drug, it can be usefully used as a pharmaceutical composition and health functional food for preventing or treating inflammatory diseases.

1 is an HPLC chromatogram graph of thistle methanol crude extract,
FIG. 2 shows the effect of luteolin 5- O- β-D-glucopyranoside compounds isolated from thistle outpost by Western blot method on iNOS and COX-2 expression in RAW 264.7 cells. It's about photography.

The present invention Maki thistle ( Cirsium) for achieving the above object maackii ) A pharmaceutical composition for the prophylaxis or treatment of inflammatory diseases with crude polysaccharide or nonpolar solvent soluble extract as an active ingredient, which has nitric oxide production in RAW 264.7 macrophages and anti-inflammatory activity of carrageenan induced mice will be.

The thistle crude extract includes water, ethanol, lower alcohols such as methanol, butanol, or a mixed solvent thereof, preferably extracts soluble in methanol.

The thistle non-polar solvent soluble extract is a non-polar solvent selected from dichloromethane, chloroform or ethyl acetate, preferably, extracted with dichloromethane and ethyl acetate.

Hereinafter, the present invention will be described in detail.

Crude extract from milk thistle of the invention, the base Theo Lin 5- O -β-D- glucopyranoside having the structure of Formula 1 below is separated from the non-polar solvent soluble extract therefrom and (luteolin 5- O -β-D- glucopyranoside ) (Hereinafter referred to as 'luteolin 5-glucopyranoside') The compound can be obtained by the following method.

(Formula 1)

The milk thistle crude extract of the present invention is powdered by grinding thistle, and then a volume of water, methanol, ethanol and butanol, which is about 3 to 20 times, preferably about 3 to 5 times, the dry weight of thistle, or These are mixed solvents having a mixing ratio of about 1: 0.1 to 1:10, preferably about 0.5 hours to 2 days at an extraction temperature of 20 to 100 ° C, preferably 20 to 70 ° C with methanol, preferably 1 After extracting by extraction method such as hot water extraction, cold needle extraction, reflux cooling extraction or ultrasonic extraction for 1 to 5 times, preferably 3 times in succession for a period of time to 1 day, filtered under reduced pressure and the filtrate was Concentrated under reduced pressure at 100 to 100 ℃, preferably 40 to 70 ℃ to obtain a crude thistle extract available in water, lower alcohol or a mixed solvent thereof.

The crude thistle extract is suspended in water and then extracted with a solvent in the order of dichloromethane, ethyl acetate, n -butanol to obtain a soluble extract of thistle nonpolar solvent of the present invention, preferably a dichloromethane soluble fraction, More specifically, thistle crude extract, that is, thistle methanol extract, may be mixed with dichloromethane: water: methanol in a ratio, preferably 10: 9: 1 to obtain a dichloromethane soluble fraction and a water soluble fraction. Ethyl acetate was added to the water soluble fraction to obtain ethyl acetate soluble fraction and water soluble fraction. Finally, the water soluble fraction was extracted with butanol to obtain butanol soluble fraction and water soluble fraction.

To the non-polar solvent ethyl acetate fractions, a mixed solvent of dichloromethane: methanol, preferably a mixed solvent of dichloromethane: methanol 1: 1, was subjected to kiesel gel column chromatography and recrystallization at a constant volume per hour, preferably 1000 ml. The method can be performed to give the luteolin 5-glucopyranoside compound.

The present invention can isolate and purify crude extracts, nonpolar solvent soluble extracts and lutein 5-glucopyranoside compounds separated therefrom from thistles.

The present invention provides nitric oxide production of lipid polysaccharide-derived RAW 264.7 macrophages containing the milk thistle extract obtained above and the lutein 5-glucopyranoside compound isolated therefrom and the right foot inflammation of carrageenan-induced mice. Provides a dietary supplement with a diet.

Pharmaceutical Compositions and Food Products Showing Lipid Polysaccharide Induced RAW 264.7 Macrophage Production and Macrophage Inflammatory Inhibitory Activity of Carrageenan Induced Mice of the Milk Thistle Extract and the Lutein 5-Glucopyranoside Compound Isolated Therefrom Composition To investigate its efficacy as a composition, lipids against luteolin 5-glucopyranoside isolated from thistle milk using nitric oxide production of lipid polysaccharide-derived RAW 264.7 macrophages and right foot inflammation in carrageenan-induced mice Nitrogen oxide production of polysaccharide-induced RAW 264.7 macrophages and inhibition of right foot inflammation in carrageenan-induced mice showed that thistle extract according to the present invention or the luteolin luteolin 5-glucopyranoside compound isolated from Lipid Polysaccharide-induced Nitric Oxide Production in RAW 264.7 Macrophages and the Progeny of Carrageenan-induced Mice It was confirmed that indicated the inflammation inhibitory activity.

Thistle extract of the present invention or the luteolin 5-glucopyranoside compound isolated therefrom has little toxicity and side effects, so can be used safely even for long-term use for the purpose of prevention.

In another aspect, the present invention is the production of nitric oxide and carrageenan-induced mouse carcinoma induction of lipid polysaccharide-derived RAW 264.7 macrophages using thistle extract obtained from the production method or luteolin 5-glucopyranoside compound isolated therefrom as an active ingredient Provides a dietary supplement showing inhibitory activity.

In addition, thistle extract of the present invention or lutein 5-glucopyranoside compound isolated therefrom is a pharmaceutical composition and health function showing the nitric oxide production of lipid polysaccharide-derived RAW 264.7 macrophages and the inhibitory activity of right foot foot in carrageenan-induced mice It can be used as a food composition.

In this case, the health functional food additive may be added to the health functional food by immersing, spraying or mixing the food, and the ratio of such additives is generally selected in the range of 0.01% _to 10%.

The health functional food is a fruit, vegetable, dried or cut products of fruit or vegetables, fruit juice, vegetable juice, mixed juices or chips, noodles, livestock processed food, fish processed food, dairy food, fermented milk food, cereals It includes any one or more of food, microbial fermented food, bakery, condiments, meat processing, acidic beverages, licorice, herbs.

Hereinafter, the present invention will be described in detail by way of examples. However, the following Examples and Experimental Examples are merely to illustrate the present invention, the contents of the present invention is not necessarily limited to the following Examples and Experimental Examples.

Example 1 Preparation of Thistle Outpost Crude Extract

Thistle outpost used in the present invention was collected from Ulsan Gyeongbuk, Gyeongbuk. Thistle outpost was shaded and crushed to obtain crude extract. 2.0 kg of ground thistle powder, 2.5 liters of methanol was added, and reflux-cooled extraction was repeated three times at regular intervals (12h, 6h, 3h) at 70 ° C., followed by filtration under reduced pressure with Wattman (USA), and then The extract was obtained by removing methanol at 40 ° C. with a vacuum rotary concentrator to obtain 450.0 g of thistle crude extract, 2 g of which were taken from nitric oxide production of lipid polysaccharide-derived RAW 264.7 macrophages and the right side of carrageenan-induced mice. It was used as a sample for the hog foot inhibitory activity.

Example 2 Preparation of Soluble Extract of Dichloromethane of Milk Thistle Outpost

2 l of dichloromethane was added to 2 l of the water-soluble fraction of thistle starch obtained in Example 1, followed by mixing 3-4 times to obtain 2 L of water-soluble fraction and 2 L of dichloromethane-soluble fraction. The fractions were dried and 125.0 g of dichloromethane soluble extract was obtained and used as a sample, and 1 g of the fraction was used as a sample for screening for nitric oxide production inhibitory activity of lipid polysaccharide-derived RAW 264.7 macrophages.

Example 3 Preparation of Milk Thistle Outpost Ethyl Acetate Soluble Extract

2 liters of ethyl acetate were added to 2 liters of the water-soluble fraction of thistle starch obtained in Example 2, followed by mixing three to four times to obtain 2 liters of the water-soluble fraction and 2 liters of the ethyl acetate soluble fraction, and then the ethyl acetate fraction. After drying, 72.0 g of ethyl acetate soluble extract was obtained and used as a sample, 1 g of which was used as a sample for screening for nitric oxide production inhibitory activity of lipid polysaccharide-derived RAW 264.7 macrophages.

Example 4. Thistle n -Preparation of Butanol Soluble Extract

2 liters of butanol was added to 2 liters of the water-soluble fraction of thistle starch obtained in Example 3, followed by mixing 3-4 times to obtain 2 liters of water-soluble fraction and 2 liters of butanol-soluble fraction, and then the water-soluble fraction and n- butanol-soluble fraction. Was dried to obtain 58.5 g of the soluble extract and n- butanol soluble extract, and 1 g of the extract was used as a sample for screening for nitric oxide production inhibitory activity of lipid polysaccharide-derived RAW 264.7 macrophages.

Example 5 Isolation of the Compound of Luteorin 5-Glucopyranoside

71.0 g of the ethyl acetate soluble extract of thistle starch of Example 3 was separated by a kielel gel column chromatography and recrystallization using a mixed solvent of dichloromethane: methanol 20: 1 → 1: 1 as a developing solvent. As a result of analyzing the compound with various analyzers such as NMR and MS, it was confirmed that the luteolin 5-glucopyranoside compound having the structure of Chemical Formula 1 below.

(Formula 1)

Appearance of substance: Yellow powder

Molecular Formula of Material: C ₂₁ H ₂₀ O ₁₁

¹ H-NMR (400 MHz, DMSO- d ₆ ) δ: 10.97 (1H, s), 9.83 (1H, s), 9.39 (1H, s), 7.38 (1H, dd, J = 9.0, 2.0 Hz), 7.36 (1H, d, J = 2.0 Hz), 6.88 (1H, d, J = 9.0 Hz), 6.79 (1H, d, J = 2.2 Hz), 6.70 (1H, d, J = 2.2 Hz), 6.55 ( 1 H, s), 5.75 (1 H, s). ¹³ C-NMR (100 MHz, DMSO- d ₆ ) δ: 176.95, 162.59, 161.37, 158.68, 158.32, 149.26, 145.69, 121.52, 118.56, 116.01, 113.12, 108.25, 105.70, 104.52, 104.33, 98.20, 77.56, 75.62 , 73.65, 69.70, 60.86.

Experimental Example 1. Lipid polysaccharide-induced inhibitory activity of nitric oxide production in crude thistle extract and fractions RAW 264.7 macrophages

end. Cytotoxicity measurement

In order to measure nitric oxide production inhibitory activity of crude polysaccharide-derived RAW 264.7 macrophages of crude extracts and fractions of thistles, cytotoxicity was first examined. Toxicity measurements on RAW 264.7 cells were analyzed by MTT assay method. RAW 264.7 cells were seeded at 1.0 × 10 ⁴ cells per well in a 96-well plate, incubated for 24 hours, exchanged with FBS-free DMEM, and the samples were treated by concentration. After 24 hours of incubation, 100 μl of MTT solution (0.5 mg / ml in PBS) was added and incubated for 2 hours. After incubation, the culture solution was removed and 100 μl of DMSO was added to dissolve the resulting crystals and the absorbance was measured at 540 nm using a microplate read spectrophotometer (Molecular Devices, VERSA max, CA, USA). Relative cytotoxicity was evaluated using the control group as 100%, and the results are expressed as the mean ± standard deviation ( n = 3).

I. LPS Induced NO  Measure

RAW 264.7 cells were aliquoted to 1.0 × 10 ⁵ cells per well in a 24-well plate and incubated for 24 hours. After exchange with FBS-free DMEM, samples of various concentrations were pretreated for 2 hours, and then incubated for 18 hours by treatment with LPS (1.0 μg / ml). NO was measured in the form of NO ₂ present in the cell culture solution using a Greries reagent. Take 100 μl of the cell culture supernatant, transfer to a 96-well plate and add 100 μl of Greries reagent at 540 nm with a microplate read spectrophotometer (Molecular Devices, VERSA max, CA, USA) Absorbance was measured. A standard calibration curve was prepared with sodium nitrite to calculate the concentration of nitrite, and AMT, an iNOS inhibitor, was used as a control. The results are expressed as mean ± standard deviation ( n = 3).

As shown in Table 1 below, the treatment of various concentrations of thistle methanol extracts with LPS-induced RAW 264.7 cells showed high NO production inhibitory activity (IC ₅₀ = 28.18 μg / mL). In the solvent fraction fraction of methanol extract, the ethyl acetate (EtOAc) fraction showed the highest inhibitory activity as 59.51% at 10 μg / mL compared to the other fractions. The NO production inhibitory activity of each fraction at the concentration of 10 μg / mL was found in the order of ethyl acetate>dichloromethane> n -butane> water fractions, and their IC ₅₀ values were as follows:

3.93 ± 0.38, 9,97 ± 0.25, 11.19 ± 1.77 and 30.55 ± 1.74 μg / mL.

sample IC _{50 (} μg / ml)  Thistle Outpost Crude Extract 28.18 ± 1.36  Thistle Outpost Dichloromethane Soluble Fraction 9.97 ± 0.25  Thistle Outpost Ethyl Acetate Soluble Fraction  3.93 ± 0.38  Thistle Outpost Butanol Soluble Fraction  11.19 ± 1.77  Thistle Outpost Water Soluble Fraction 30.55 ± 1.74  Reference drug (AMT) 1.04 0.02

Experimental Example 2 Lipid Polysaccharide Induction of Lutein 5-Glucopyranoside Compound Isolated from Thistle RAW  264.7 Suppressing Nitric Oxide Production in Macrophages

Lipid polysaccharide-induced nitric oxide generation inhibitory activity of the lipid polysaccharide-derived RAW 264.7 macrophages obtained in Example 5 was carried out in the same manner as in Experimental Example 1, and the results are shown in Table 2 below. Indicated.

As shown in Table 2 below, the luteolin 5-glucopyranoside compound showed an IC ₅₀ value of 64.36 ± 0.05 μM and the control compound AMT showed an IC ₅₀ of 0.08 ± 0.00 μM.

Serial number sample IC _{50 (} μM)  One Luteolin 5-Glucopyranoside 64.36 ± 0.05 Control group AMT 0.08 ± 0.00

Experimental Example  3. Thistle Crude extract and  Isolated Luteolin  5- Glucopyranoside  Carrageenan Induction of Compounds in Mice Right foot  Edema inhibitory activity

To assess anti-inflammatory activity in vivo , the method of Winter et al . (1962) was slightly modified to measure λ-CGN-induced paw edema in mice. Four-week-old male ICR mice (20 g) were purchased from Samtaco Bio Korea Ltd. Korea, where temperature (22 ± 2 ° C), relative humidity (50 ± 10%), and contrast cycle (light: In the animal breeding room where the dark cycle) was adjusted to be 12 hours apart, normal breeding and water were ingested freely for 7 days, and preliminary breeding was performed when the body weight was 27 ± 1 g. The group was divided into groups of seven animals and intraperitoneally administered 0.05 mL of the compound dissolved in DMSO. After 1 hour, 1% CGN dissolved in saline was administered 0.05 mL of the left hind foot to induce foot edema. After 5 hours, the volume of the edema was measured using a plethysmometer (Ugo Basil, Italy), and the volume of the foot that was initially increased compared to the volume of the foot that was not treated with the compound was regarded as edema. Indomethacin (indomethacine) was used as a control.

Edema Inhibition Rate (%) = [(CGN-Treatment Control-Experimental Group) / (CGN-Treatment Control-Control Except CGN)] × 100

As shown in Table 3 below, thistle crude extract and the luteolin 5-glucopyranoside compound were intraperitoneally administered 250 mg / kg and 100 mg / kg, respectively, to inhibit edema, inhibiting 59.34% and 48.31%. It showed a corresponding anti-inflammatory activity with the compound indomethacin (inhibition rate of 60.43%).

Serial number sample % Inhibition rate One Thistle Outpost Crude Extract 250 mg / kg 59.34 2 Luteolin 5-Glucopyranoside 100 mg / kg 48.31 Control group Indomethacin 60.43

Experimental Example  4. Thistle Leaf Crude extract  And Fraction RAW  264.7 t-butyl hydro in macrophages Peroxide  Judo Active oxygen species ( t - butylhydroproxide induced ROS ) Inhibitory activity

Determination of t- butylhydroproxide induced ROS inhibitory activity in RAW 264.7 macrophages of crude extracts and fractions of thistle leaves was carried out in a well- plated 96-well black plate. Wells were aliquoted to 2.0 × 10 ⁴ cells and incubated for 24 hours.

Serum-free RPMI-1640 was exchanged, and the samples were treated for each concentration and incubated for 1 hour. After treatment with t- BHP (final concentration 200 μM), incubation for 30 minutes with addition of DCFH-DA (20 μM), and the fluorescence intensity of the oxidized DCF was measured using a microplate fluorescence reader (Bio-). Tek Instruments Inc., FLx 800, Winooski, UT, USA) were used to measure excitation and emission wavelengths at 485 and 530 nm, respectively. The relative ROS content was evaluated using the control group as 100%, and the results are expressed as the mean ± standard deviation ( n = 3).

t- BHP disrupts the redox state in the cell, producing reactive oxygen species such as alkoxyl radicals and hydroxyl radicals (Elliott, SJ, Eskin, SG, Schilling, WP Effect). of t -butyl-hydroperoxide on bradykinin-stimulated changes in cytosolic calcium in vascular endothelial cells.J . Biol. Chem., 264, 38063810. 1989). Therefore, cells were treated with t- BHP to induce oxidative stress. ROS production was measured using DCFH-DA (2 ', 7'-dichlorodihydrofluorescein diacetate) (Lebel and Bondy, 1990), a ROS-sensitive fluorescent indicator. When DCFH-DA is treated in living cells, it enters the cell and is deacetylated by the action of the esterase enzyme present in the cell, which is converted into DCFH (2 ', 7'-dichlorodihydrofluorescein), which is converted into ROS and The reaction was quantitatively converted into fluorescent DCF (2 ', 7'-dichlorofluorescein) and measured by fluorescence spectrometer. As shown in Table 4 below, treatment of various concentrations of thistle methanol extract with t- BHP-induced RAW 264.7 cells showed high ROS production inhibitory activity (IC ₅₀ = 7.51 μg / mL). In the fractions solvent-distilled from the methanol extract, the EtOAc fraction showed the highest inhibitory activity as 68.32% at a concentration of 10 μg / mL compared to the other fractions. At 10 μg / mL concentration, ROS production inhibitory activity of each fraction was shown in the order of ethyl acetate> n -butane>dichloromethane> water fractions, and their IC ₅₀ values are shown in Table 4 below.

sample IC _{50 (} μg / ml)   Thistle Leaf Crude Extract 7.51 ± 0.60   Thistle leaf dichloromethane soluble fraction   11.22 ± 0.77   Thistle leaf ethyl acetate soluble fraction   1.48 ± 0.08   Thistle leaf butanol soluble fraction  2.59 ± 0.25   Thistle Leaf Water Soluble Fraction  21.28 ± 1.74   Reference drug (Tororox)  14.04 ± 1.27

Experimental Example  5. Thistle From outpost  Isolated Luteolin  5- Glucopyranoside  Of RAW 264.7 Cells Of Compound iNOS Wow COX -2 effect on expression

end. Western Western blot  through iNOS  And COX -2 expression analysis

In order to measure the expression level of iNOS and COX-2 by LPS, RAW 264.7 cells were pretreated with various concentrations of compounds for 2 hours, and then treated with LPS (1.0 μg / ml) for 18 hours. After washing twice with cold PBS (phosphate buffered saline) and scraping off, the cells were lysed by lysis buffer (lysis buffer) at 4 ° C. for 30 minutes. Intracellular protein was obtained by centrifugation at 14,000 × g for 20 minutes at 4 ° C. The concentration of protein was quantified by the Bradford method. Proteins were mixed with gel buffer (gel buffer, Bio-Rad), boiled for 5 minutes, denatured, and the same amount of protein was electrophoresed using 10% SDS-polyacrylamide gel. After electrophoresis, transfer to PVDF membrane using a wet transfer system (Bio-Rad, Hercules, Calif., USA), and the membrane is blocked in order to inhibit nonspecific binding of antibodies. 5% (w / v) non-fat dry milk in Tris-buffered saline containing 0.1% Tween-20, pH 7.4 (TBST buffer)) was incubated for 1 hour at room temperature. Thereafter, the membrane was reacted with iNOS and an antibody against COX-2 (1: 1000 in TBST buffer) at 4 ° C. overnight. iNOS and COX-2 expression levels were reacted with HRP-attached anti-rabbit IgG (1: 2000 in TBST buffer) for 1 hour at room temperature, followed by X-signaling using Supersignal West Pico Chemiluminescent Substrate. It was confirmed by exposure to a ray film. Molecular weights were identified using full-range rainbow molecular weight markers (Amersham). Western blotting data (Western blotting data) was confirmed three times more than the same trend.

Luteorin 5- O -glucoside did not show any cytotoxicity up to 200 μM concentration. Perhaps the introduction of glucoside at the C-5 position of luteolin was likely to reduce cytotoxicity. These results are in good agreement with previous studies on NO production inhibition experiments in RAW 264.7 cells of Flavonoid compounds; Lu Theo Lin 7- O - glucoside was seed-toxic base lower than Theo Lin (Hu, C., Kitts, DD , Luteolin and luteolin-7-O-glucoside from dandelion flower suppress iNOS and COX-2 in RAW 264.7 cells. Mol. Cell.Biochem., 265, 107-113. 2004). Also, Caco-2 intestinal cells, Hu and Kitts, 2004). However, IC ₅₀ values for inhibition of NO production of luteolin and luteolin 7-glucoside were similar at 8.7 μM and 9.4 μM (Hu and Kitts, 2004). The IC ₅₀ value of luteolin 5-glucoside is 64.36 μM, which is unusual in that the NO production inhibitory activity is much lower than that of luteolin 7-glucoside, while the cytotoxicity is low. Therefore, it is believed that the hydroxyl group at the C-5 position glucosylates the NO production inhibitory activity of LPS-induced RAW264.7 cells by structural differences. Expression of iNOS and COX-2 proteins is associated with inflammatory responses (Yun et al ., 1996). Inhibition of iNOS and COX-2 expression of luteolin 5- O -glucoside was found for the first time (see FIG. 2). These results indicate that luteolin 5- O -glucoside inhibits NO and ROS production through iNOS and COX-2 expression in RAW264.7 cells. Flavone and flanol compounds inhibit iNOS and COX-2 protein expression by inhibiting mRNA expression and transcription (Chen, YC, Shen, SC, Lee, WR, Hou, W. C, Yang, LL, Lee, TJF, Inhibition of nitric oxide synthase inhibitors and lipopolysaccharide induced NOS and cycloxygenase-2 gene expression by rutin, quercetin and quercetin pentaacetate in RAW 264.7 macrophages.J. Cell Biochem ., 82, 537-548. 2001). Treatment with 20 μM of luteolin and luteolin 7-glucoside inhibits iNOS and COX-2 expression without cell damage (Hu and Kitts, 2004). On the other hand, many flavonoid compounds are known to affect only COX-2 activity without affecting iNOS activity (Liang, YC, Huang, YT, Tsai, SH, Lin-Shiau, SY, Chen, CF, Lin, JK). , Suppression of inducible cyclooxygenase and inducible nitric oxide synthase by apigenin and related flavonoids in mouse mcrophages. Carcinogenesis, 20, 1945-1952. 1999).

For reference, Figure 1 is a chromatogram showing the HPLC analysis of the methanol crude extract for each thistle part. 1, peak 1 is lutein 5-glucopyranoside (Rt: 28.2 min), peak 3 is apigenin 7-glucoside (Rt: 32.6 min), peak 8 is luteolin (Rt: 41.2 min) , Peak 9 is apigenin (Rt: 44.6 min).

HPLC analysis conditions of the crude extract for each thistle part are as follows.

JASCO HPLC system (Tokyo, Japan)

Analysis program: Borwin chromatographic system (Le Fontanil, France)

Column: Phenomenex C18 Reversed Phase Column (Phenomenex, 4.6 250 mm, 5 m)

Temperature: 25 ℃

UV detector: 340 nm

Solvent condition: A solvent: 0.5% phosphoric acid solution, B solvent: 100% methanol, A: B mixed solvent (75%: 25%) for 60 minutes at 0%: 10,000% at a flow rate of 0.5 ml / min.

In the HPLC results, the peaks 1 and 8, which are the main peaks, were identified as lutein 5-glucopyranoside and luteolin, respectively, and quantitatively contained 5.67% of luteolin 5- O -glucoside. From these results, it could be estimated that this compound is composed of major compounds in the crude methanol extract.

And, Figure 2 relates to a photograph measuring the effect on the iNOS and COX-2 expression of RAW 264.7 cells of lutein 5-glucopyranoside compound isolated from thistle outpost by Western blot method The effects of luteolin 5-glucopyranoside compounds on the expression of iNOS and COX-2, NF-κB-related inflammatory responses, were examined by Western blot method. These protein expressions increased during LPS treatment was confirmed to be concentration-dependently inhibited by luteolin 5-glucoside pretreatment.

1: Luteolin 5-glucopyranoside 3: Apigenin 7-glucosi
8: luteolin 9: apigenin

Claims (5)

(1) drying and grinding the thistle into powder;
(2) Methanol was added to the milk thistle powder prepared in step (1), and reflux-cooled extraction was performed three times at intervals of 12 hours, 6 hours, and 3 hours, respectively, at a temperature of 70 ° C., followed by filtration under reduced pressure, followed by 40 to 70 Concentrating under reduced pressure at ℃ to obtain a crude methanol extract; And
(3) dissolving the crude methanol extract obtained by the above step (2) in distilled water, dichloromethanol mixture was repeated three times, and then concentrated and dried under reduced pressure at 40 to 70 ℃ to obtain a dichloromethanol extract Manufactured, including;
A method for preparing an extract for preventing or treating inflammatory diseases, which comprises a 5-O-β-D-glucopyranoside compound represented by Formula 1 as an active ingredient.
[Chemical Formula 1]

The method of claim 1,
(4) The dichloromethanol extract obtained by the above step (3) was dissolved in distilled water, and ethyl acetate was mixed to repeat the above three times, and the mixture was concentrated under reduced pressure and dried at 40 to 70 ° C. to obtain an ethyl acetate extract. The method of producing an extract for preventing or treating inflammatory diseases further comprising.
3. The method of claim 2,
(5) The ethyl acetate extract obtained by the above step (4) was dissolved in distilled water, n -butanol was mixed, and the above-described partitioning was repeated three times. Concentration and drying under reduced pressure at 40 to 70 ° C. extracted n -butanol. It further comprises the step of obtaining, a method of producing an extract for preventing or treating inflammatory diseases.
The method of claim 1,
In the step (3), dichloromethane: water: methanol in a ratio of 10: 9: 1 to prepare a method for producing an extract for preventing or treating inflammatory diseases, characterized in that used.
The method according to any one of claims 1 to 4, characterized in that it has nitric oxide production of lipid polysaccharide-induced RAW 264.7 macrophages and inhibitory activity of right foot paw of carrageenan-induced mice, Therapeutic extracts.

Images