KR101781083B1 - Composition comprising compounds isolated from Salicornia herbacea for preventing or treating vascular inflammation or sepsis - Google Patents

Abstract

The present invention relates to a composition for preventing or treating vascular inflammation or sepsis comprising a compound derived from Hamcho, wherein the compound is flavanones or chromone derivatives, wherein the compound is HMGB1 (high mobility group box 1) and inhibits the mortality of mice according to the CLP procedure (cecal ligation and puncture operation), thereby preventing or treating vascular inflammation or sepsis or preventing or improving vascular inflammation or sepsis It is easily available as a health functional food.

Description

TECHNICAL FIELD The present invention relates to a composition for preventing or treating vascular inflammation or sepsis, which contains a compound isolated from green tea,

The present invention relates to a composition for preventing or treating vascular inflammation or sepsis which contains a compound isolated from green tea.

The vascular endothelium plays an important role in a variety of physiological functions including vasomotor tone, blood transport, hemostasis, proliferation, survival, congenital or adaptive immunity, and the like. Thus, vascular endothelial dysfunction results in physiological response abnormalities, which are mainly induced from a variety of vascular inflammatory conditions such as acute lung injury, anaphylaxis, atherosclerosis, sepsis (Pharmacological Reports. 2008, 60 Journal of Cellular Physiology, 2014, 229, 620-630).

Sepsis (sepsis) is caused by toxins produced by microorganisms or microorganisms when they are infected with microorganisms such as Pseudomonas aeruginosa, Escherichia coli, Streptococcus, Staphylococcus aureus, and Pneumococcus, and it causes chills, systemic symptoms such as high fever, joint pain, headache, State that appears. If these symptoms are severe, hypotension is accompanied by decreased urine volume and septic shock (Nat. Med. 2003, 9, 517-524). Although the infection pathways of microorganisms are not well known, appendicitis, otitis media, skin pyrexia, pressure ulcer, lung disease, cholecystitis, pyelonephritis and osteomyelitis are known as the cause of sepsis. Blood tests, urine tests, and CSF tests can be used to diagnose sepsis. Increased leukocyte counts and acute inflammatory agents also help to diagnose sepsis.

Until now, the fundamental treatment for sepsis has not been confirmed yet. Sepsis is not well treated with conventional anti-inflammatory therapy and even the only FDA-approved drotrecogin alfa (Xigris ^® , Engl. J. Med. 2012, 366, 2055-2064) The effect of the treatment is unclear. Currently, sepsis is treated mainly by injection of antibiotics or antifungal drugs, and the duration of the treatment is determined by the type of microorganism. Hemodialysis or transfusion may also be performed depending on the patient's condition. When antibiotics and antifungal agents are well tolerated, sepsis may be cured, but patients may die because they are difficult to treat when they are infected with a drug resistant microorganism, when their immunity is weak, or when they start treatment too late. In addition, the mortality rate of sepsis is very high, reaching 50-70%, and it is known that it accounts for a high percentage of deaths worldwide (Nat. Med. 2003, 9, 517-524). Complications associated with sepsis can lead to sequelae. Complications include meningitis, neurological sequelae, and pyogenic arthritis together with bone growth abnormalities may occur.

The official name of Salicornia herbacea is Tungtung Madi, but it is mainly called Hamcho. It grows in tidal flats where the waters are well and relatively firm. The stem is fleshy and has a cylindrical shape. The branches are opposite to each other, and the degenerated scaly leaves are running against each other, and the nodes bulge out, resulting in the name Tungtungmadi. It is distributed all over the west coast and south coast of Korea. Green tea has long been eaten for many years, cutting the stem, boiling the soup, or grind it together with flour and knead it together to eat. Because it is rich in minerals, it is known as a health food. It removes toxins and succumbs that are accumulated in the body and has excellent therapeutic effect on cancer, uterine myoma, sinusitis, hypertension, hypotension, back pain, diabetes, bronchial asthma, hypothyroidism, hyperthyroidism, skin disease, arthritis, It is known as the amazing herb that it has. It is also used for treatment of diseases caused by inflammation and arthritis, such as eliminating sukbyeo and treating obesity, improving blood circulation, clearing blood, strengthening blood vessels, and so on. The effect is excellent.

The green tea contains phytosterol, phenolic acid, flavonoid, polysaccharide, dicaffeoyl quinic acid, procatechuic acid, ferulic acid, , Triterpenoid saponin, and the like. Particularly, among the active ingredients of green tea, 3-caffeoyl-4-dihydrocafeoylquinic acid isolated from green tea has been reported to have an antioxidant effect, gout treatment, anti-inflammatory effect, obesity and fatty liver.

HMGB1 (high mobility group box 1) is a protein expressed in the endothelium in the presence of vascular injury such as sepsis (Toxicol. Appl. Pharm. 2012, 262, 91-98; J. Cell. Physio. 2013, 228, 975 (Science, 1999, 285, 248-251), the inventors of the present invention found that flavanone or chromone derivative compounds derived from Hamcho exhibited inhibition of the expression of HMGB1, Inflammatory or sepsis, and thus the present invention has been completed.

Korean Patent Publication No. 2005-0087418 (pharmaceutical composition for antiinflammation containing 3-caffeoyl-4-dihydrocappaheylquinic acid isolated from Hamcho), published Aug. 31, 2005)

Aird, W. C. Endothelium in health and disease. Pharmacological Reports. 2008, 60, 139-143. Bae, J. S. et al., Activated protein C inhibits high mobility group box 1 signaling in endothelial cells. Blood. 2011, 118, 3952-3959. Choi, Y. H. et al., Rhododendric acid A, a novel ursane-type PTP1B inhibitor from the endangered plant Rhododendron brachycarpum G. Don., Bioorg. Med. Chem. Lett. 2012, 22, 6116-6119. Deanfield, J. E. et al., Endothelial function and dysfunction: Testing and clinical relevance. Crirculation. 2007, 115, 1285-1295. Lee, J. D. et al., Flavonol-rich RVHxR from Rhus verniciflua Stokes and its major compounds, fisetin inhibits inflammation-related cytokines and angiogenic factors in rheumatoid arthritic fibroblast-like synovial cells and in vivo models. Int. Immunopharmacol. 2009, 9, 268-276. Lee, W. et al., Endocan Elicits Severe Vascular Inflammatory Responses in Vitro and in Vivo. Journal of Cellular Physiology. 2014, 229, 620-630. Lee, W. et al., Barrier protective effects of withaferin in HMGB1-induced inflammatory responses in both cellular and animal models. Toxicol Appl. Pharmacol. 2012, 262, 91-98. Matsumori, N. et al., Stereochemical determination of acyclic structures based on carbon-proton spin-coupling constants. A method of configuration analysis for natural products. J. Org. Chem. 1999, 64, 866-876. Riedemann, N. C. et al., Novel strategies for the treatment of sepsis., Nat. Med. 2003, 9, 517-524. Ranieri, V. M. et al., Drotrecogin alfa (activated) in adults with septic shock. Engl. J. Med. 2012, 366, 2055-2064. Yang, E.J. et al., Barrier protective effects of rosmarinic acid on HMGB1-induced inflammatory responses in vitro and in vivo. J. Cell. Physio. 2013, 228, 975-982. Wang, H. et al., HMGB-1 as a late mediator of endotoxin lethality in mice. Science. 1999, 285, 248-251. Antioxidant, Cytotoxic and Anti-inflammatory Activity of Kangsumi, Green Tea and Green Tea Seed Extract, Seoul Women's University, 2011, Food Engineering.

It is an object of the present invention to provide a composition for preventing or treating vascular inflammation or sepsis which contains a compound isolated from green tea.

The present invention relates to 7,2'- dihydroxy-6-methoxy -2 S of formula (I) - flavanone (Compound 1), 2'-hydroxy-6,7-methoxy -2 S-flavanone ( Compound 2), 5,2'-dihydroxy-6,7-methylenedioxyflavanone (Compound 3), 7-hydroxy-6,8-dimethoxychromone (Compound 4), 6-methoxychroma (Compound 5) and 6,7-dimethoxychromone (Compound 6). The present invention also relates to a composition for preventing or treating vascular inflammation or sepsis.

[Chemical Formula 1]

These compounds are flavanones or chromone derivatives derived from Hamcho and have an effect of inhibiting the expression of HMGB1 (high mobility group box 1).

The invention of 7,2'- dihydroxy-6-methoxy -2 S Formula 1-flavanone (Compound 1), 2'-hydroxy-6,7-methoxy -2 S-flavanone ( Compound 2), 5,2'-dihydroxy-6,7-methylenedioxyflavanone (Compound 3), 7-hydroxy-6,8-dimethoxychromone (Compound 4), 6-methoxychroma (Compound 5) and 6,7-dimethoxychromone (Compound 6). The present invention also provides a health functional food for preventing or ameliorating vascular inflammation or sepsis. The health functional food may be selected from the group consisting of various foods, beverages, gums, tea and vitamin complex.

The present invention relates to novel compounds 7,2'- dihydroxy-6-methoxy -2 S of the general formula (2) to 4-flavanone (Compound 1), 2'-hydroxy-6,7-methoxy -2 S -Flavanone (Compound 2) or 7-hydroxy-6,8-dimethoxychromone (Compound 4).

(2)

(3)

[Chemical Formula 4]

Accordingly, the present invention is 7,2'- dihydroxy-6-methoxy -2 S from Salicornia extract - flavanone (Compound 1), 2'-hydroxy-6,7-methoxy -2 S-flavanone (Compound 2) and 7-hydroxy-6,8-dimethoxychromone (Compound 4).

Hereinafter, the present invention will be described in detail.

The invention of 7,2'- dihydroxy-6-methoxy -2 S Formula 1-flavanone (Compound 1), 2'-hydroxy-6,7-methoxy -2 S-flavanone ( Compound 2), 5,2'-dihydroxy-6,7-methylenedioxyflavanone (Compound 3), 7-hydroxy-6,8-dimethoxychromone (Compound 4), 6-methoxychroma (Compound 5) and 6,7-dimethoxychromone (Compound 6). The present invention also relates to a composition for preventing or treating vascular inflammation or sepsis.

The compound may be flavanones or chromone derivatives, which are derived from a green tea extract. The green tea extract may be obtained by extracting green tea with water, a lower alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof. The extraction conditions are not limited, but it is preferable that the water, the lower alcohol having 1 to 4 carbon atoms, or the mixed solvent thereof is added 1 to 20 times as much as the weight of ammonia, and the extraction temperature is preferably 20 to 100 ° C , And the extraction time is preferably from 1 hour to 7 days. The above-mentioned green tea extract may be prepared by a known method which is used for extractive separation of plant components, such as extraction with water or various organic solvents, distribution of organic solvent and water, and column chromatography, May be fractionated or purified and used.

Preferably, the green tea extract is obtained by concentrating green tea extract obtained by extracting and concentrating green tea with water, a lower alcohol having 1 to 4 carbon atoms or a mixed solvent thereof, adding 1 to 50 times of water to the weight of the green tea extract, suspending the green tea extract, And a green tea fraction obtained by adding a solvent selected from the group consisting of hexane, chloroform, ethyl acetate, and butanol to water. The green tea fraction is preferably a concentrate of a hexane layer obtained by suspending a green tea extract obtained by extracting and concentrating green tea with water, a lower alcohol having 1 to 4 carbon atoms or a mixed solvent thereof, and then mixing with hexane, The concentrate of the chloroform layer obtained by removing the layer (chloroform) from the remaining residue (water layer) or the concentrate of the ethyl acetate layer obtained by mixing the residue (water layer) remaining after removing the chloroform layer with ethyl acetate, A concentrate of the butanol layer obtained by removing the layer and mixing butanol with the remaining residue (water layer), or a concentrate of the residue (water layer) remaining after removing the butanol layer. The compounds of formula (I) of the present invention are flavanones or chromone derivatives which are separated from green tea extract or green tea leaf fraction. In addition, other conditions for the fractionation are not limited, but it is preferable to add water to the green tea extract to 1 to 50 times the weight of the green tea extract to prepare a suspension. Then, the same amount of hexane, chloroform, ethyl acetate, Butanol can be added to the reaction mixture. Further, chloroform is added to the remaining residue after removing the hexane layer, chloroform layer is removed, and ethyl acetate is added to the remaining residue. When ethyl acetate is removed and butanol is added to the remaining residue, the same amount Each solvent (chloroform, ethyl acetate or butanol) can be added sequentially to fractionation. Each fractionation time at this time is not particularly limited, but is preferably within 10 minutes to one day.

Chromatography can be used to separate the fenugreek extract or to isolate the flavanone or chromone derivative of the present invention. The chromatography can be carried out by silica gel column chromatography, Lewis-20 column chromatography (LH-20 column chromatography), ion exchange resin chromatography, medium pressure liquid chromatography (MPLC), thin layer chromatography (TLC), silica gel vacuum liquid chromatography silica gel vacuum liquid chromatography, reversed phase chromatography, and high performance liquid chromatography.

The green tea extract can be extracted from any part selected from a certain part such as plantago, root, leaf, stems of green tea plant.

On the other hand, the present invention relates to novel compounds 7,2'- dihydroxy-6-methoxy -2 S - flavanone (Compound 1), 2'-hydroxy-6,7-methoxy -2 S - flavanone ( Compound 2) or 7-hydroxy-6,8-dimethoxychromone (compound 4), and provides a method for separating the novel compounds from green tea. Preferably, the novel compounds 7,2'- dihydroxy-6-methoxy -2 S - flavanone (Compound 1), 2'-hydroxy-6,7-methoxy -2 S - flavanone (Compound 2) or 7-hydroxy-6,8-dimethoxychromone (Compound 4) is prepared by suspending the green tea extract obtained by extracting and concentrating green tea with water, a lower alcohol having 1 to 4 carbon atoms or a mixed solvent thereof, A hexane layer obtained by mixing with hexane, an ethyl acetate layer obtained by removing the hexane layer and mixing ethyl acetate in the residue (water layer) remaining, and a concentrate of the residue (water layer) remaining after removing the ethyl acetate layer .

The present invention is 7,2'- dihydroxy-6-methoxy -2 S of formula (I) - flavanone (Compound 1), 2'-hydroxy-6,7-methoxy -2 S - Plastic (Compound 2), 5,2'-dihydroxy-6,7-methylenedioxyflavanone (Compound 3), 7-hydroxy-6,8-dimethoxychromone (Compound 4) (5) and 6,7-dimethoxychromone (Compound (6)). The present invention also provides a pharmaceutical composition for preventing or treating vascular inflammation or sepsis.

The pharmaceutical compositions may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories and sterilized injection solutions according to conventional methods. 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 suppository bases include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like.

The dosage of the pharmaceutical composition containing the flavanone or chromone derivative compound of the present invention will depend on the age, sex, and weight of the subject to be treated, the specific disease or condition to be treated, the severity of the disease or condition, . 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. These compounds may be added to the pharmaceutical composition of the present invention in an amount of preferably 0.001 to 90% by weight, more preferably 0.001 to 50% by weight, and most preferably 0.001 to 30% by weight.

The pharmaceutical composition of the present invention can be administered to mammals such as rats, livestock, humans, and the like in 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 of the present invention has little toxicity and side effects, it can be safely used even for long-term administration for preventive purposes.

The present invention is 7,2'- dihydroxy-6-methoxy -2 S of formula (I) - flavanone (Compound 1), 2'-hydroxy-6,7-methoxy -2 S - Plastic (Compound 2), 5,2'-dihydroxy-6,7-methylenedioxyflavanone (Compound 3), 7-hydroxy-6,8-dimethoxychromone (Compound 4) Prevention or amelioration of vascular inflammation or sepsis, including at least one compound selected from the group consisting of ethoxychromanone (Compound 5) and 6,7-dimethoxychromone (Compound 6), and a pharmaceutically acceptable food additive additive And a health functional food. The above compounds may be added to the health functional food of the present invention in an amount of preferably 0.001 to 90% by weight, more preferably 0.001 to 50% by weight, and most preferably 0.001 to 30% by weight. The health functional food of the present invention includes forms such as tablets, capsules, pills, and liquids. Examples of the foods to which the extract of the present invention can be added include various foods, beverages, gums, tea, vitamins , And health functional foods.

The present invention relates to a composition for preventing or treating vascular inflammation or sepsis comprising a compound derived from Hamcho, wherein the compound is flavanones or chromone derivatives, wherein the compound is HMGB1 (high mobility group box 1) and inhibits the mortality of mice according to the CLP procedure (cecal ligation and puncture operation), thereby preventing or treating vascular inflammation or sepsis or preventing or improving vascular inflammation or sepsis It is easily available as a health functional food.

On the other hand, Korean Patent Laid-Open No. 2005-0087418 discloses that 3-caffeoyl-4-dihydrocappa oil quinic acid derived from Hamcho has an anti-inflammatory effect. [Antioxidant, cytotoxic And anti-inflammatory activity, Seoul Women's University, 2011, Food Science and Technology] In addition, the anti-inflammatory activity of the green tea extract is also disclosed. However, the anti-inflammatory substance described above may be said to have an inhibitory effect on vascular inflammation or sepsis none. In addition, in the present invention, since the activity of flavanone or chromone derivative derived from green tea, which is not a green tea extract, is confirmed, and since these compounds are also compounds having different chemical structures from 3-caffeoyl-4-dihydrocappaheinquinic acid, The effects of the compounds of the present invention on the vascular inflammation inhibitory effect or the sepsis treatment and on the effects of the green tea extract or 3-caffeoyl-4-dihydrocafeoylquinic acid are completely different.

Fig. 1 shows the cytotoxicity of the compounds 1 to 6 of the present invention through MTT assays.
2 is a graph showing the concentration of the compounds 1 to 6 represented by [占 퐉 / mouse] in the animal test of the present invention in terms of [占 퐂 / mouse].
FIG. 3 shows the results of ELISA confirming the effect of the compounds 1 to 6 of the present invention inhibiting HMGB1 expression in a sepsis cell model (FIGS. 3A and 3B) or an animal model of sepsis (FIG. 3C and 3D), respectively.
FIG. 4 shows the results of confirming that the compounds 1 to 6 of the present invention inhibit vascular permeability due to damage of vascular endothelial cells in the sepsis cell model (FIG. 4A) or the sepsis animal model (FIG. 4B), respectively.
FIG. 5 shows the expression of VCAM-1, ICAM-1 and E-selection (FIG. 5A) and the cell surface marker receptor TLR2 (Toll-like receptor 2) Inhibition of TLR4 (Toll-like receptor 4) and RAGE expression (Fig. 5B) by ELISA.
FIG. 6 shows experimental results showing that the compounds 1 to 6 of the present invention inhibit vascular inflammation. FIG. 6A shows that the compounds 1 to 6 of the present invention inhibit the migration of monocytes to vascular endothelium following vascular inflammation FIG. 6B shows that the compounds 1 to 6 of the present invention inhibit the adhesion of monocytes to vascular endothelium due to vascular inflammation, and FIG. 6C shows that the compounds 1 to 6 of the present invention induce HMGB1 or CLP Inhibited the migration of leukocyte from vascular endothelium to the vascular endothelium.
7 shows that compounds 1-6 of the present invention inhibit the expression of TNF-? (FIG. 7A), IL-1? (FIG. 7B) and phosphorylated NF-? B (phospho-NF-KB, FIG. 7C) It shows the result of confirmation.
Figure 8 shows the survival rate of sepsis animals treated with the compounds 1-6 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  One. From Hamcho  Separation of compounds &gt;

8.3 kg of aerial aerials of dried Salicornia herbacea were extracted with 72 L of methanol for 4 days at room temperature. After repeating this one more time, the resulting liquid phases were collected, filtered and concentrated to give a brown slurry of methanol extract 1.9 kg).

All of the methanol extracts were suspended in water (4.0 L), and then sequentially fractionated with n -hexane (3 L portions were repeated 4 times), ethyl acetate (3 L portions were added 4 times) and water. Thus, n- hexane fraction (106.6 g) and ethyl acetate fraction (45.7 g) were obtained.

The ethyl acetate fraction (45.7 g) was separated by silica gel column VLC (25 x 18 cm) and eluted with n- hexane: acetone (100: 0 to 0: 100 [100: 0, 90:10, 80:20, , 60:40, 50:50, 30:70, 10:90, 0: 100], 10% [v / v] increment, each step 2ℓ, final washing with 6ℓ of 100% MeOH) E2 (1.40 g), E4 (1.30 g), E5 (1.22 g), E6 (1.55 g), E7 (2.80 g), E8 (5.40 g), E9 (9.53 g), and E10 (18.03 g)].

The small fraction E5 (2.77 g) was separated by RP-C18 (column Biotage SNAP Cartridge, KP-C18-HS, 400 g, 75 Psi Max) using MPLC and eluted with methanol: water (60% MeOH) Seven fractions [E5-1 (458 mg), E5-2 (275 mg), E5-3 (280 mg), E5-4 (220 mg), E5-5 (241 mg), E5-6 Mg) and E5-7 (600 mg)].

The small fraction E5-3 was further separated by semi-prep HPLC and eluted with methanol: water (AB, 0-10 min, 50-70% A; 10-60 min, 70-80% A; 60-70 min, 80-100% compound 1 by elution with a concentration gradient conditions (t _R: 26min, 7.6㎎) and seven small fraction [E5-3-1 (32.3㎎), E5-3-2 ( 9.5㎎), E5-3-3 ( E5-3-4 (14.4 mg), E5-3-5 (33.6 mg), E5-3-6 (15.5 mg) and E5-3-7 (4.2 mg).

The mixture of the small fractions E5-3-2 and E5-3-4 was separated by semi-prep HPLC and eluted with methanol: water (AB, 0-10 min, 50-60% A, 10-60 min, 60-68% -65min, 68-100% a) elution conditions to give the compound 2 _(R t to: obtain a 52min, 5.2㎎).

The small fraction E5-4 was subjected to semi-prep HPLC and eluted with methanol: water (AB, 0-10 min, 50-70% A, 10-50 min, 70-80% A, 50-70 min, 80-100% E5-4-1 (17.7 mg), E5-4-2 (3.2 mg), E5-4-3 (3.6 mg), E5-4-4 (8.0 mg), E5-4- 5 (10.3 mg), E5-4-6 (58.4 mg), E5-4-7 (9.0 mg) and E5-4-8 (21.9 mg).

The small fraction E5-4-4 was subjected to semi-prep HPLC under the conditions of methanol: water (AB, 0-10 min, 50-75% A, 10-50 min, 75-80% A, 50-60 min, 80-100% eluted compound 3 with (t _R: 32min, 5.7㎎) was obtained.

The small fraction E6 was separated by RP-C18 (column Biotage SNAP Cartridge, KP153 C18-HS, 400 g, 75 Psi Max) using MPLC and eluted with gradient conditions of methanol: water (30-50% MeOH, 6 L) E6-1 (121.4 mg), E6-2 (739 mg), E6-3 (175 mg), E6-4 (312.8 mg), E6-5 (258.7 mg), E6-6 ), E6-7 (90 mg), E6-8 (71.8 mg) and E6-9 (803 mg)].

The small fraction E6-2 was separated by semi-prep HPLC and eluted with methanol: water (AB, 0-10min, 25% A; 10-15min, 25-30% A; 15-20min, 30% 40% A, 25-70 min, 40% A, 70-85 min, 40-80% A, 85-90 min, 80% A, 90-100 min, 80-100% A, 100-110 min, 100% A) (E6-2-1 (3.1 mg), E6-2-2 (0.5 mg), E6-2-3 (0.4 mg) and E6-2-4 (40.5 mg) , E6-2-5 (8.7 mg), E6-2-6 (16.1 mg), E6-2-7 (16.1 mg), E6-2-8 (31.1 mg), E6-2-9 (13.3 mg) , E6-2-10 (16.7 mg), E6-2-11 (26.1 mg), E6-2-12 (11.4 mg), E6-2-13 (0.6 mg) and E6-2-14 (165.5 mg) ].

The small fraction, E6-2-8, was separated by semi-prep HPLC and eluted with a gradient of methanol: water (AB, 0-10 min: 15% A, 10-15 min: 30% A, 15-60 min: 30% A, 60-65 min: 100 % a, 65-75min: elution with gradient conditions of 100% a) to 5 (t _R compound: was obtained 18min, 3.5㎎).

Compound 4 (3.0 mg) was crystallized in a small fraction, E6-2-10, and recrystallized on methanol to give pure product.

The small fraction E6-4 (312.8 mg) was separated by semi-prep HPLC and eluted with methanol / water (AB, 0-10 min, 25% A; 10-15 min, 25-30% A; 20-40 min, 30-40% A, 25-70 min, 40% A, 70-85 min, 40-80% A, 85-90 min, 80% A, 90-100 min, 80-100 % a; 100-110min, by elution with a concentration gradient conditions of 100% a) 6 (t _R compound: to obtain a 65min, 3.5㎎).

* The percentages of the chromatographic eluant of Example 1 all represent the mixing ratio (%) mixed in [v: v].

&Lt; Example 2: Identification of physicochemical properties of a compound >

Example 2-1. 7,2'-dihydroxy-6-methoxy-2 S - flavanone (Compound 1)

7,2'-Dihydroxy-6-methoxy- 2 S -flavanone;

¹ H-NMR (600 MHz, CD ₃ OD) and ¹³ C-NMR (150 MHz, CD ₃ OD): See Table 1

- 114.42°; c 0.05, MeOH)은 황색의 분말(yellow powder)로서 분리되었다. HR-ESI-MS 분석결과 나타난 나트륨 분자이온 피크로부터 화합물 1의 분자식은 C₁₆H₁₄O₅로 결정된다([M+Na]⁺, m/z 309.1623, [calcd for C₁₆H₁₄O₅Na]). IR 스펙트럼(KBr, ν_max, ㎝^-1)은 화합물 1이 히드록실 그룹(3306㎝^-1), 카보닐 그룹(1656㎝^-1) 및 벤젠 고리(1504, 1471, 1458㎝^-1)를 갖고 있음을 나타낸다. UV-Vis 스펙트럼(MeOH, λ_max(log ε))은 330 (sh), 279 (3.94) 및 237 (4.05)nm에서 최대값을 갖는 것으로 나타낸다. 화합물 1의 ¹H NMR 및 ¹³C NMR 스펙트럼(표 1 참조)은 플라보노이드의 특징을 나타낸다. δ _H 2.82 (dd, J = 3.3, 17.0, H-3a) 및 2.90 (dd, J = 13.0, 17.0, H-3b) (δ_C, 43.9, C-3)에서의 메틸렌 그룹, δ_H 5.73 (dd, J = 3.2, 12.9, δ_C, 76.7, C-2)에서의 메틴, δ _C 193.8 (C-4)에서의 카보닐 그룹은 화합물 1이 플라바논임을 가리킨다. 화합물 1의 δ _H 5.73 (H-2) 메틴과 δ _C 155.2 (C-2')의 HMBC 상관관계 및 δ _H 6.83 (H-3'), 7.17 (H-4'), 6.91 (H-5'), 7.49 (H-6')의 COSY 상관관계로부터 B-ring의 2' 위치에 히드록시 그룹을 갖는 것으로 확인된다. HMBC 분석에서 δ _H 7.31 양성자와 δ _C 193.8의 상관관계 신호는 5번 위치에 하이드록실 그룹의 부재를 나타내고, δ _H 3.87의 메톡시 양자는 δ _C 156.9의 4차 탄소에 위치하고 있음이 확인된다. 2번 위치의 절대구조는 CD 스펙트럼에서의 235.2nm의 코튼 효과(Cotton effect)에 근거하여 S 형의 절대구조인 것을 알 수 있다. 따라서, 화합물 1은 7,2'-디히드록시-6-메톡시-2S-플라바논(7,2'-dihydroxy-6-methoxy-2S-flavanone)의 구조를 갖는 신규 화합물로서 확인된다. Compound 1 (

- 114.42 DEG; c 0.05, MeOH) was isolated as a yellow powder. From the sodium molecular ion peak shown in the HR-ESI-MS analysis, the molecular formula of Compound 1 is determined as C ₁₆ H ₁₄ O ₅ ([M + Na] ⁺ , m / z 309.1623, _{[calcd for C 16 H 14 O} 5 Na]). The IR spectrum (KBr, v _max , cm ^-1 ) shows that compound 1 has a hydroxyl group (3306 cm ^-1 ), a carbonyl group (1656 cm ^-1 ) and a benzene ring (1504, 1471, 1458 cm ^-1 ) . The UV-Vis spectra (MeOH, λ _max (log ε)) are shown to have maximum values at 330 (sh), 279 (3.94) and 237 (4.05) nm. The ¹ H NMR and ¹³ C NMR spectra of Compound 1 (see Table 1) characterize the flavonoids. _{δ H 2.82 (dd, J =} 3.3, 17.0, H-3a) and 2.90 (dd, J = 13.0, 17.0, H-3b) (δ C, 43.9, C-3) a methylene group, δ _H 5.73 in ( dd, carbonyl groups in _{methine, δ C 193.8 (C-4} ) at _{J = 3.2, 12.9, δ C} , 76.7, C-2) indicates that the compound 1 is flavanone. Compound 1 of the δ _H 5.73 _(H-2) methine, and _{δ C 155.2 (C-2 '} ) HMBC correlations and δ _H 6.83 _(H-3 of'), 7.17 (H-4 '), 6.91 (H-5 '), 7.49 (H-6') from the COSY correlation, the hydroxy group is found at the 2 'position of the B-ring. In the HMBC analysis, the correlation signal between δ _H 7.31 protons and δ _C 193.8 indicates the absence of hydroxyl groups at position 5, and the methoxy quantities of δ _H 3.87 are located at the quaternary carbon of δ _C 156.9. It can be seen that the absolute structure at position 2 is the S- type absolute structure based on the Cotton effect at 235.2 nm in the CD spectrum. Thus, Compound 1 was 7,2'- dihydroxy-6-methoxy -2 S - is identified as a novel compound having the structure of the flavanone (7,2'-dihydroxy-6-methoxy -2 S -flavanone) .

Example 2-2. 2'-hydroxy-6,7-methoxy-2 S - flavanone (Compound 2)

2'-Hydroxy-6,7-methoxy- 2 S -flavanone;

¹ H-NMR (600 MHz, CD ₃ OD) and ¹³ C-NMR (150 MHz, CD ₃ OD): See Table 1

- 146.75°; c 0.05, MeOH)는 노란색의 분말로서 수득되었다. 화합물 2의 HR-ESI-MS 데이터에 따른 분자식은 C₁₇H₁₆O₅([M+Na]⁺, m/z 323.0895 [calcd for C₁₇H₁₆O₅Na])로 확인된다. IR 스펙트럼(KBr, ν_max, ㎝^-1)은 화합물 2에 히드록실 그룹(3414㎝^-1), 카보닐 그룹(1651㎝^-1), 벤젠 고리(1502, 1455, 1438㎝^-1) 등의 특징이 있음을 보여준다. 화합물 2의 UV-Vis 스펙트럼(MeOH, λ_max(log ε))은 337 (3.85), 275 (4.10) 및 237 (4.24)에서 최대값을 갖는 것으로 확인된다. 화합물 2의 ¹H NMR 및 ¹³C NMR 데이터(표 1 참조)는 δ _H 3.81에서의 메톡시 양자를 제외하고 화합물 1과 매우 비슷하다. 화합물 2의 HMBC 상관관계는 δ _H 3.81, δ _H 3.88에서의 메톡시 양자가 δ _C 158.4 및 146.1의 4차 탄소와 관련이 있음을 나타낸다. 화합물 2의 C-2의 절대구조는 CD 스펙트럼에서의 299.6nm의 양성 코튼 효과(Cotton effect)에 기반하여 S형 구조인 것으로 확인된다. 따라서, 화합물 2는 2'-히드록시-6,7-메톡시-2S-플라바논(2'-hydroxy-6,7-methoxy-2S-flavanone)의 화학구조를 갖는 신규 화합물인 것으로 확인된다. Compound 2 (

- 146.75 [deg.]; c 0.05, MeOH) was obtained as a yellow powder. The molecular formula of the HR-ESI-MS data for compound 2 is identified as _{C 17 H 16 O 5 ([} M + Na] +, m / z 323.0895 [calcd for C 17 H 16 O 5 Na]). The IR spectrum (KBr, 僚_max , cm ^-1 ) was obtained by adding a hydroxyl group (3414 cm ^-1 ), a carbonyl group (1651 cm ^-1 ), a benzene ring (1502, 1455, 1438 cm ^-1 ) . The UV-Vis spectrum (MeOH,? _Max (log?)) Of Compound 2 is found to have a maximum value at 337 (3.85), 275 (4.10) and 237 (4.24). ¹ H NMR and ¹³ C NMR data of Compound 2 (see Table 1) is very similar to Compound 1 except for the methoxy proton at δ _H 3.81. The HMBC correlation of compound 2 shows that the methoxy quantities at δ _H 3.81 and δ _H 3.88 are related to the quaternary carbons of δ _C 158.4 and 146.1. The absolute structure of C-2 of compound 2 is confirmed to be an S-type structure based on the positive Cotton effect of 299.6 nm in the CD spectrum. Thus, Compound 2 is 2'-hydroxy-6,7-methoxy -2 S - identified to be a novel compound having a chemical structure of the flavanone (2'-hydroxy-6,7-methoxy -2 S -flavanone) do.

Examples 2-3. 5,2'-dihydroxy-6,7-methylenedioxyflavanone (Compound 3)

5,2'-Dihydroxy-6,7-methylenedioxyflavanone;

¹ H-NMR (600 MHz, CD ₃ OD) and ¹³ C-NMR (150 MHz, CD ₃ OD): See Table 2

NMR and MS analysis showed that Compound 3 was identified as 5,2'-dihydroxy-6,7-methylenedioxyflavanone (5,2'-dihydroxy-6,7-methylenedioxyflavanone).

Examples 2-4. 7-hydroxy-6,8-dimethoxychromone (Compound 4)

7-Hydroxy-6,8-dimethoxychromone;

¹ H-NMR (600 MHz, CDCl ₃ ) and ¹³ C-NMR (150 MHz, CDCl ₃ ): See Table 1

Compound 4 was obtained as a white crystalline compound. The molecular formula according to the HR-ESI-MS data of Compound 4 is confirmed by C ₁₁ H ₁₀ O ₅ ([M + Na] ⁺ , m / z 245.0426 [calcd for C ₁₁ H ₁₀ O ₅ Na]). ¹ H NMR and ¹³ C NMR data for compound 4 is very similar, except for oxidized oleic pinik quaternary presence of carbon, and chromones (chromone) compound at δ _C 135.0. The HMBC correlation of compound 4 shows that one methyl proton at δ _H 7.30 is related to the carbonyl carbon at δ _C 176.8 (C-4), confirming it is H-5. This means that position 8 of compound 4 (position 8) was substituted. It also means that the two methoxy groups at δ _H 3.92 (s) and δ _H 3.98 (s) are related to the quaternary carbon of δ _C 145.9 (C-6) and 135.0 (C-8). Accordingly, it is confirmed that Compound 4 is a novel compound having a structure of 7-hydroxy-6,8-dimethoxychromone.

Examples 2-5. 6-methoxychromanone (Compound 5)

6-Methoxychromanone;

¹ H-NMR (600 MHz, CD ₃ OD) and ¹³ C-NMR (150 MHz, CD ₃ OD): See Table 2

NMR and MS analysis showed that Compound 5 It is identified as 6-methoxychromanone.

Example  2-6. 6,7- Dimethoxychromone (Compound 6)

6,7-Dimethoxychromone;

¹ H-NMR (600 MHz, CD ₃ OD) and ¹³ C-NMR (150 MHz, CD ₃ OD): See Table 2

NMR and MS analysis confirmed that Compound 6 was 6,7-dimethoxychromanone.

Position Compound 1 Compound 2 Compound 4 ¹ H ¹³ C ¹ H ¹³ C ¹ H ¹³ C 2 5.73
(dd, J = 12.9, 3.2) 76.7 5.74
(d, J = 2.5) 76.9 7.79
(d, J = 5.9) 154.5 3 2.82
(dd, J = 17.0,3.3)
2.90
(dd, J = 17.0, 13.0) 43.9 2.80
(d, J = 2.3) 43.4 6.23
(d, J = 5.9) 112.3 4 - 193.8 - 193.8 - 176.8 5 7.31 (s) 108.1 7.27 (s) 107.9 7.30 (s) 99.5 6 - 145.2 - 146.1 - 145.9 7 - 156.9 - 158.4 - 144.1 8 6.48 (s) 104.7 6.65 (s) 101.6 - 135.0 9 - 160.6 - 160.6 - 146.6 10 - 113.7 - 114.1 - 117.8 One' - 127.2 - 127.0 - - 2' - 155.2 - 155.3 - - 3 ' 6.83
(d, J = 8.1) 116.2 6.83
(dd, J = 8.1, 0.7) 116.2 - - 4' 7.17
(dt, J = 7.8, 1.5) 130.2 7.17
(td, J = 7.9, 1.6) 130.3 - - 5 ' 6.91 (m) 120.7 6.89
(dt, J = 7.6, 0.7) 120.7 - - 6 ' 7.49
(d, J = 7.7) 127.6 7.51
(dd, J = 7.7,1.4) 127.7 - - 6-OCH ₃ 3.87 (s) 56.6 3.83 (s) 56.6 3.92 (s) 56.6 7-OCH ₃ - - 3.89 (s) 56.7 - - 8-OCH ₃ - - - - 3.98 (s) 61.7 -OCH ₂ O- - - - - - - - The chemical shifts (δ) are in ppm from TSM.
- The coupling constant J values (in hertz) are given in parentheses.
- All assignments are based on the COZY, HMQC, and HMBC experiment.

Position Compound 3 Compound 5 Compound 6 ¹ H ¹³ C ¹ H ¹³ C ¹ H ¹³ C One - - - - - - 2 5.67 dd (3.0, 13.3) 76.6 3.95 t (6.2) 58.9 8.12 d (5.9) 156.8 3 2.79 dd (3.0, 17.2)
2.98 dd (13.3, 17.2) 43.1 3.18 t (6.2) 41.7 6.32 d (5.9) 112.6 4 6.14 h 199.4 - 199.7 - 179.2 5 - 91.4 7.58 m 111.9 7.49 s 104.83 6 - 157.7 - 149.1 - 149.6 7 - 130.4 7.58 m 124.7 - 157.6 8 - 146.2 6.88 d (8.2) 115.8 7.12 s 101.2 9 - 161.4 - 153.4 - 154.6 10 - 105.1 - 130.6 - 118.7 6-OCH ₃ - - 3.92 s 56.4 3.93 h 56.6 7-OCH ₃ - - - - 3.97 s 57.0 8-OCH ₃ - - - - - - One' - 126.5 - - - - 2' - 155.3 - - - - 3 ' 6.84 m 116.2 - - - - 4' 7.13 td (1.6, 8.0) 129.2 - - - - 5 ' 6.87 m 120.7 - - - - 6 ' 7.42 dd (1.4, 7.7) 127.6 - - - - -OCH ₂ O- 5.95 dd (3.0, 13.3) 103.8 - - - -

< Example  3. Cytotoxicity>

(3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) assay was performed to confirm that the compounds of the present invention were cytotoxic. HUVEC (Human umbilical vein endothelial cells), and then a frequency divider in a 96-well plate per well 5 × 10 ³ dogs, 24 hours later, ESM (effective screening medium) was replaced with culture medium (HUVEC cells Cambrex Bio Science (Charles City, IA, USA) and incubated with EBM-2 basal media and growth supplement (Cambrex Bio Science) supplied thereto. At this time, the compounds 1 to 6 of the present invention were treated to a final concentration of 1 to 20 μM, and after 48 hours, 100 μl of a 1 mg / ml MTT solution was added to the cells and allowed to react for 4 hours. After that, 150 mu l of DMSO (dimethyl sulfoxide) was added to dissolve the formazan salt produced in the cells. The amount of the formazan salt was measured at 540 nm using a microplate reader (Tecan Austria GmbH, Austria) The results are shown in Fig.

Referring to FIG. 1, it is confirmed that all of the compounds 1 to 6 of the present invention are not cytotoxic.

< Example  4. Induction of sepsis animal models>

Sepsis was induced in mice using a cecal ligation and puncture operation that exploded the mouse's cecum. The mice used for CLP surgery were male C57BL / 6 mice, and 10 mice were used per group (6 ~ 7 weeks old and 18 ~ 20g mice were purchased and adapted for 12 days in a laboratory. After using Orient Bio Co Sungnam, Kyungki-Do, Republic of Korea). Mice were anesthetized with zoletil 50 and 3% (w / v) isoflurane (Forane®, Choongwae Pharma. Corp., Seoul, Korea). To induce a sepsis model, 2 cm of the abdomen was incised to expose the cecum and the nearby intestines. The cecum 5 mm from the end of the cecum was tightly bound with a 3.0-silk suture, and the cecum was punctured with a 22-gauge needle. The cecum was gently squeezed to allow a small amount of excreta (intestinal package) to leak through the puncture site. The excreta was exposed to the abdominal cavity and the open area was sealed with a 4.0-silk suture. Control groups (sham, sham-operated mice) were simply stabbed and re-stitched without performing the step of tying and bursting the cecum. Each mouse was exposed to sepsis symptoms 24 hours after CLP surgery, with symptoms such as shivering, bristled hair or weakness.

< Example  5. In the sepsis model HMGB1  Expression confirmation>

Example  5-1. Sepsis cell model HMGB1  Confirmation of expression

HUVECs were cultured in 6-well plates and treated with LPS (lipopolysaccharide, 100 ng / ml) for 4 hours (control group treated with saline) For 6 hours.

In a 96-well plastic flat microtiter plates (Corning, NY, USA), 0.02% sodium azide was added to perform competitive enzyme-linked immunosorbent assays (HUVEC) HMGB1 protein (Abnova) dissolved in 20 mM carbonate / bicarbonate buffer (pH 9.6) was coated and reacted overnight at 4 ° C for 18 hours or more. After that, the plate was washed 3 times with PBS-T (PBS-0.05% [w / w] Tween 20), then PBS-T was added and kept overnight at 4 ° C for 18 hours or more.

HMGB1 protein (for Abnova, dilution in PBS-T for protein determination) and HUVEC conditioned culture media (including LPS-treated sample) were incubated with anti-HMGB1 antibody (Abnova, PBS-T At 1: 1000 dilution) at 37 &lt; 0 &gt; C for 90 minutes. The HUVEC cell extracts contained 1 mM PMSF, 1 mM Na ₃ VO ₄ , 1 mM NaF, 1 μg / ml aprotinin, 1 μg / ml pepstatin, 1 μg / ml leupeptin and 10% The supernatant was obtained by centrifugation (15000 rpm, 4 ° C, 20 minutes) after decomposition by vortexing at 4 ° C for 1 hour using a protein separation reagent containing RIPA dissolution buffer (Upstate Biotechnology, USA) .

The cell culture of HMGB1 protein, HUVEC, which had been reacted with the antibody, was transferred to a plate coated with HMGB1, and reacted at room temperature for 30 minutes. Each plate was then washed three times with PBS-T and reacted with secondary antibodies (peroxidase-conjugated anti-rabbit IgG antibodies, 1: 2000 dilution in R & D Systems, PBS-T) for 90 minutes at room temperature.

Each plate was washed three times with PBS-T and treated with 200 μl of substrate solution (100 μg / ml o- phenylenediamine & 0.003% H ₂ O ₂ ) for 60 minutes in a dark room at room temperature. Next, 50 μl of 8N H ₂ SO ₄ was added to stop all the reactions, and the absorbance was confirmed at 490 nm. The analysis results are shown in FIGS. 3A and 3B.

3A and 3B, it is confirmed that the compounds of the present invention in the sepsis cell model have the effect of inhibiting the expression of HMGB1 remarkably in the HUVEC treated with LPS.

Example  5-2. Sepsis HMGB1  Confirmation of expression

Mice treated with CLP according to the method of Example 4 were intravenously administered with Compound 1 to 6 (1-10 μM / mouse) of the present invention (administered 12 hours after performing CLP surgery).

The values of [μM / mouse] and [μg / mouse] of each compound used in the animal experiment are shown in FIG.

After 24 hours of CLP, the mice were sacrificed and blood was collected and centrifuged at 2000 xg for 5 minutes to obtain serum samples. The ELISA procedure was performed in the same manner as in Example 5-1, and the analysis results are shown in FIGS. 3C and 3D.

3C and 3D, it was confirmed that the compounds 1 to 6 of the present invention significantly inhibited the expression of HMGB1 in the sepsis animals induced by CLP surgery.

< Example  6. Vascular cell  Confirmation of permeation inhibiting effect>

Example  6-1. In the sepsis cell model Vascular cell  Confirm permeation inhibiting effect

Compounds 1 to 6 (10 μM) of the present invention were treated with HUVEC to confirm that each compound restored HMGB1-induced vascular wall damage (resulting in permeability due to microperforation of blood vessels). For this purpose, the flow of Evans blue-bound albumin in the cell layer cultured using a 2-compartment chamber model (Blood 2011, 118, 3952-3959) was quantitatively measured.

HUVECs were cultured for 3 days in a 12 mm diameter transwell with a pore size of 3 [mu] m at 5 x 10 &lt; ⁴ &gt; / well. When cells were filled in each transwell, LPS (100 ng / ml, 4 hours) or HMGB1 (1 μg / ml, 16 hours) was treated and reacted, and each compound was treated for 6 hours. Each transwell well was then washed with PBS (pH 7.4) and 0.5 ml of Evans blue solution (0.67 mg / ml) diluted in cell culture medium containing 4% (w / v) BSA was prepared. Fresh cell culture medium was added to the lower chamber, and the upper chamber was replaced with the above Evans blue solution. After 10 minutes, the OD (optical density) of the lower chamber was measured at 650 nm and the results are shown in FIG. 4A.

4A, it can be confirmed that the compounds 1 to 6 of the present invention all inhibit the permeability (flow of Evans blue solution) due to cell damage, and thus, it is excellent in inhibiting vascular inflammation in the HMGB1-induced sepsis cell model .

Example  6-2. In an animal model of sepsis Vascular cell  Confirm permeation inhibiting effect

Male C57BL / 6 mice were prepared (10 mice per group, 6-7 weeks old and 18-20g mice were purchased and adapted for 12 days in a laboratory, using Orient Bio Co., Sungnam, Kyungki-Do, Republic (2 μg / mouse) of Evans Blue solution and 1% (w / v) Evans Blue solution dissolved in saline solution at 6 hours after intravenous injection of each sample (10 μM / mouse) Followed by intravenous injection.

Two hours after the intravenous injection of Evans Blue solution, the mice were sacrificed and the peritoneal exudate was collected by centrifugation at 200 xg for 10 minutes while the abdominal cavity was washed with 5 ml of saline solution. The absorbance of the supernatant of the centrifuge was confirmed at 650 nm. This is because the dye of the Evans blue solution flows into the abdominal cavity and can confirm the permeability due to the microperforations in the vascular cells (Int. Immunopharmacol. 2009, 9, 268-276.). The results are shown in FIG. 4B.

Referring to FIG. 4B, it can be seen that there is a large amount of dye leakage in the HMGB1-treated mouse. In the group treated with the compounds 1 to 6, the leakage of the dye is remarkably suppressed in the mice. Thus, it can be shown that the compounds inhibit sepsis by protecting the vascular wall damage.

< Example  7. Cell adhesion molecule  And cell surface Marker  Confirmation of receptor expression>

The expression of proteins related to cell adhesion such as VCAM-1 (vascular cell adhesion molecule-1), ICAM-1 (intercellular adhesion molecule-1) and E-selection was confirmed by whole-cell ELISA.

When cells were confluent at 90%, HMGB1 (1 / / ml) was incubated in HUVEC cultured on a 96-well plate for 16 hours, and the compounds 1 to 6 (10 쨉 M) of the present invention were treated for 6 hours. The cells were then washed with PBS (phosphate buffered saline), and the cells were fixed with 50 μl of 1% (w / v) paraformaldehyde for 15 min at room temperature of 25 ° C . After removing the 1% (w / v) paraformaldehyde and washing the cells, a mouse anti-human monoclonal antibody (VCAM-1, ICAM-1 or E-selection) USA, 1:50 dilution) was added. After 1 hour, each cell was washed 3 times and 100 μl of 1: 2000 peroxidase-conjugated anti-mouse IgG antibody (1: 2000 peroxidase-conjugated anti-mouse IgG antibody, Sigma, Saint Louis, MO) , Washed three times, and reacted with o- PPD substrate ( o- phenylenediamine substrate, Sigma, St. Louis, Mo.). The absorbance for the resulting color reaction was measured at 490 nm and is shown in Figure 5A. Toll-like receptor 2 (TLR2), TLR4 (Toll-like receptor 4) and RAGE (Receptor for advanced glycation end products) receptors, which are cell surface markers expressed when vascular inflammation occurs in vascular endothelial cells Expression was also confirmed for each marker using the A-9, H-80 and A-9 antibodies (Santa Cruz, CA) in the same manner, the results of which are shown in FIG. 5B.

5A and 5B, Compounds 1 to 6 of the present invention inhibited cell adhesion molecules VCAM-1, ICAM-1 and E-selection (FIG. 5A) and cell surface marker receptor TLR2 (Toll-like receptor 2), TLR4 (Toll-like receptor 4) and RAGE (FIG. 5B), and thus the compounds are shown to exhibit excellent vascular inflammation inhibitory effects.

< Example  8. Identification of monocyte and leukocyte migration into vascular endothelium following vascular inflammation>

Example  8-1. Identification of monocyte migration into vascular endothelium following vascular inflammation

To confirm the migration of monocytes to vascular endothelium following vascular inflammation, a 6.5 mm diameter transwell plate with a filter having a pore size of 8 mu m was used. HUVEC (6 x 10 &lt; ⁴ &gt; / cells) was cultured in the lower chamber below the filter of the transfer well plate for 3 days, and the cell culture plate was filled with cells. Monocyte THP-1 cells were also cultured in the upper chamber of the plate layer where the HUVECs were cultured. HUVECs were treated with HMGB1 (1 μg / ml) before the THP-1 cells were placed in the upper chamber (THP- Maintained in RPMI 1640 medium (containing 10% FBS [v / v], 55 μM 2-mercaptoethanol) containing L-glutamine at a density of 2 × 10 ⁵ to 1 × 10 ⁶ cells / HMGB1 was reacted with HUVEC for 16 hours, and compounds 1 to 6 (10 μM) of the present invention were treated for 6 hours. Then, the cells in the upper chamber that did not move under the filter were removed, and the mononuclear cells migrated on the filter in the lower chamber side were fixed with 8% (w / v) glutaraldehyde, v) 0.25% (w / v) crystal violet solution dissolved in an aqueous methanol solution. The results of the staining were visually confirmed using an optical microscope and the degree of migration of mononuclear cells with reference to the migration index was shown in FIG. 6A.

Referring to FIG. 6A, it was confirmed that the compounds 1 to 6 of the present invention significantly inhibited the migration of monocytes to vascular endothelium following vascular inflammation.

Example  8-2. Confirmation of monocyte adhesion to vascular endothelium due to vascular inflammation

To confirm adhesion of mononuclear cells to vascular endothelial cells, THP-1 cells (1.5 × 10 ⁶ / ml, 200 μl / well) were labeled with Vybrant DiD dye and then added to HUVEC cells.

For this, HUVEC was first reacted with HMGB1 (1 / / ml) for 16 hours, and the compounds 1 to 6 (10 쨉 M) of the present invention were treated for 6 hours. Subsequently, THP-1 cells labeled with Vybrant DiD dye were added and incubated at 37 ° C for 1 hour. Unadsorbed cells were washed with cell culture medium and the degree of adhesion of HUVEC and THP-1 cells was measured by fluorescence microscopy The results are shown in Fig. 6B.

Referring to FIG. 6B, it was confirmed that Compounds 1 to 6 of the present invention remarkably inhibited the adhesion of monocytes to vascular endothelium due to vascular inflammation.

Example  8-3. Identification of leukocyte migration into vascular endothelium

In order to measure the migration of leukocytes, Compound 1 to 6 (10 μM / mouse) of the present invention was intravenously injected into mice, and HMGB 1 (2 μg / mouse) was intravenously injected after 6 hours.

In addition, the compounds 1 to 6 of the present invention (10 μM / mouse) were intravenously administered (after 12 hours after the CLP surgery) to the mice that had undergone CLP surgery by the method of Example 4.

After the peritoneal effusion was collected, 20 μl of the peritoneal effusion was mixed with 0.38 ml of Turk 'solution (0.01% crystal violet in 3% acetic acid) And the number of white blood cells identified through the eye was visually observed. The results are shown in Fig. 6C.

Referring to FIG. 6C, it can be confirmed that the compounds 1 to 6 of the present invention were inhibited from leukocyte migration to vascular endothelium from HMGB1 or CLP-induced mice, and the number of leukocytes in vascular endothelium was remarkably reduced.

< Example  9. Expression of inflammatory factors>

Compounds 1 to 6 (10 μM) of the present invention were treated with HUVEC for 6 hours, and then treated with HMGB1 (1 μg / ml, 16 hours) for reaction. The expression of each inflammatory factor was confirmed using an ELISA kit, and the results are shown in FIG.

TNF-? And IL-1? (R & D Systems, Minneapolis, MN, USA), Total-NF-κB and phospho-NF-κB (# 7174, # 7173, Cell Signaling Technology, Danvers, MA, USA).

7, the compounds 1 to 6 of the present invention inhibit the expression of TNF-α, IL-1β and phosphorylated NF-κB (phospho-NF-κB) .

< Example  10. Determine survival rate in sepsis animal models>

Compounds 1 to 6 of the present invention (10 μM / mouse) were intravenously administered twice (12 hours and 50 hours after CLP surgery, twice in total) to the mice that had undergone CLP surgery according to the method of Example 4 20 per group). Survival of the mice was confirmed from 6 hours to 126 hours after performing CLP surgery (observed according to Kaplan-Meier survival analysis), and the survival results of the mice over time are shown in FIG. 8 .

Referring to FIG. 8, it was found that the mouse group administered with the compounds 1 to 6 of the present invention had a survival rate of 40 to 60% at 126 hours after the CLP surgery in all the compounds, indicating that the treatment effect of sepsis was excellent have.

&Lt; Example 11: Toxicity test &

Example 11-1. Acute toxicity

(7,2'-dihydroxy-6-methoxy-2 S -flavanone) or compound 4 (7-hydroxy-6,8-dimethoxychromone) of the present invention in a short period of time (Within 24 hours) to determine toxicity to the animal body, and to determine the mortality rate. An ICR mouse line, a common mouse, was assigned to each group of 10 mice. In the control group, only the PEG-400: Tween-80: ethanol (8: 1: 1, v: v: 8: 1: 1, v: v: v). After 24 hours of administration, the respective mortality rates were examined. As a result, it was confirmed that mice were all alive in the control group and the test group to which Compound 1 or Compound 4 of the present invention was administered at a concentration of 2 g / kg / day.

Example 11-2. Organ organs toxicity test in experimental group and control group

The long-term toxicity test was carried out using the compound 1 (7,2'-dihydroxy-6-methoxy-2 S -flavanone) or the compound 4 (7-hydroxy-6,8-dimethoxychromone) C57BL / 6J mice (10 mice per group) for 8 weeks. (8: 1: 1, v: v: v) alone or in combination with a test group to which Compound 1 or Compound 4 of the present invention was administered and PEG-400: Tween-80: ethanol After 8 weeks from the control animals, the blood was collected and the concentration of glutamate-pyruvate transferase (GPT) and blood urea nitrogen (BUN) in the blood was 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, followed by a general tissue section preparation, histological observation with an optical microscope, and no abnormalities were observed in all tissues.

&Lt; Formulation Example 1 >

Formulation Example 1-1. Manufacture of tablets

200 g of Compound 1 (7,2'-dihydroxy-6-methoxy-2 S -flavanone) or Compound 4 (7-hydroxy-6,8-dimethoxychromone) of the present invention was dissolved in 175.9 g of lactose, 180 g of potato starch and 32 g of colloidal silicic acid. A 10% (w / v) gelatin solution was added to the mixture, followed by pulverization 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 present compound (7,2'-dihydroxy-6-methoxy-2 S -flavanone) or compound 4 (7-hydroxy-6,8-dimethoxychromone), 0.6 g of sodium chloride and 0.1 g of ascorbic acid was 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

The compound 1 (7,2'-dihydroxy-6-methoxy-2 S -flavanone) or the compound 4 (7-hydroxy-6,8-dimethoxychromone) By weight to prepare a cooking sauce for health promotion.

Formulation Example 2-2. Manufacture of flour food products

0.1 wt% of Compound 1 (7,2'-dihydroxy-6-methoxy-2 S -flavanone) or Compound 4 (7-hydroxy-6,8-dimethoxychromone) And the mixture was used to prepare breads, cakes, cookies, crackers and noodles to prepare foods for health promotion.

Preparation Example 2-3. Manufacture of soups and gravies

The compound 1 (7,2'-dihydroxy-6-methoxy-2 S -flavanone) or the compound 4 (7-hydroxy-6,8-dimethoxychromone) % &Lt; / RTI &gt; by weight to prepare soup for health promotion and juices.

Formulation Example 2-4. Manufacture of dairy products

(7,2'-dihydroxy-6-methoxy-2 S -flavanone) or Compound 4 (7-hydroxy-6,8-dimethoxychromone) of the present invention was added to milk in an amount of 0.1% , 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 Compound 1 (7,2'-dihydroxy-6-methoxy-2 S -flavanone) or Compound 4 (7-hydroxy-6,8-dimethoxychromone) To 1,000 ml of carrot juice, vegetable juice for health promotion was prepared.

Formulation Example 2-6. Manufacture of fruit juice

0.1 g of Compound 1 (7,2'-dihydroxy-6-methoxy-2 S -flavanone) or Compound 4 (7-hydroxy-6,8-dimethoxychromone) To 1,000 ml of grape juice, fruit juice for health promotion was prepared.

Claims (6)

(Compound 4), 6-methoxychromanone (Compound 5), and 6,7-dimethoxychromone (Compound 6) of the following Formula 1 are selected from the group consisting of 7-hydroxy-6,8-dimethoxychromone Or a pharmaceutically acceptable salt thereof, as an active ingredient.
[Chemical Formula 1]

The method according to claim 1,
Wherein said compound has an effect of inhibiting the expression of HMGB1 (high mobility group box 1). (Compound 4), 6-methoxychromanone (Compound 5), and 6,7-dimethoxychromone (Compound 6) of the following Formula 1 are selected from the group consisting of 7-hydroxy-6,8-dimethoxychromone Or a pharmaceutically acceptable salt thereof, as an active ingredient.
[Chemical Formula 1]

The method of claim 3,
Wherein said health functional food is selected from the group consisting of various foods, beverages, gums, tea and vitamin complex. delete Extracting Salicornia herbacea with water, C1 to C4 lower alcohol or a mixed solvent thereof to prepare a green tea extract; Sequentially fractionating the green tea extract with hexane and ethyl acetate; And separating 7-hydroxy-6,8-dimethoxychromone (Compound 4) of the following formula (2) by chromatography on each of the above fractions.
(2)

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