KR101655180B1 - Composition comprising extract of Rhododendron brachycarpum or megastigmane glucosides isolated therefrom for preventing or treating sepsis - Google Patents

Abstract

The present invention relates to a composition for preventing or treating septicemia, which contains a moxa agar extract or a megastigue main glycoside isolated therefrom, wherein the composition is effective for inhibiting the expression of HMGB1 (high mobility group box 1) in vascular endothelial cells Can be easily used as a health functional food for preventing or treating sepsis or for preventing or treating sepsis.

Description

[0001] The present invention relates to a composition for preventing or treating septicemia, which comprises extracts of Rhododendron or Rhododendron main meandering gypsum isolated therefrom,

The present invention relates to a composition for preventing or treating septicemia which contains a mexican plant extract or a megastigue main glycoside isolated therefrom.

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 the systemic symptoms such as fever, joint fever, 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.

Rhododendron brachycarpum is an evergreen shrub of azalea ( Azacaceae ) of the dicotyledonous plant. It grows in high mountains of Korea (Jiri, Ulleungdo, Gangwon and northern regions) and Japan. The height is 1 to 4 m, and the bark is white with ashy. Leaves are alternate but 5 ~ 7 are gathered at the end of the branch, and they are elliptical or elliptical, and they are leather (leather-like texture). Length 8 ~ 20cm, width 2 ~ 5cm, edge is flat and dried back. It is dark green on the outside and light brown hairs on the back. The petiole is 1 ~ 3 cm long. The flowers bloom in June ~ July, and run on the ends of 10 ~ 20 branches with guns. Small peduncle is reddish brown with dense hair. The corolla is a funnel-shaped, white or pale yellow, with green spots on the inside and cracked into five branches. There are 10 stamens and 1 pistil. Ovaries have dense brown hairs. The fruit is oval shape with an oval shape, about 2 cm in length and ripened brown in September. The crimson flower is called Rhododendron brachycarpum var. Roseum . It is planted in the garden for ornamental purposes, and the leaves are diuretics and tonic. Rhizoma has traditionally been used mainly for the treatment of diseases such as cardiovascular disease, diabetes, hypertension, hepatitis, rheumatoid arthritis, and headache, but the above-mentioned effects have not been studied much yet.

HMGB1 (high mobility group box 1) is a protein expressed when the blood vessel is damaged due to progression of sepsis (Toxicol. Appl. Pharm. 2012, 262, 91-98; J. Cell. Physio. 2013, 982). The present inventors completed the present invention by confirming that the compounds of the extracts of Pseudomonas sylvestris and Pseudomonas sylvestris inhibit the expression of HMGB1 and thus have a therapeutic effect on sepsis.

Korean Patent Laid-Open No. 2009-0131399 discloses that a mixed extract of Phellodendron japonica, Rhododendron japonica and Burdock is effective for antiinflammation and skin irritation mitigation. In Korean Patent No. 508402, a herbal medicine extract containing Rhodiola is administered to hepatitis It has been disclosed that the pioneer extract or the compound isolated therefrom is effective in treating sepsis.

Korean Patent Laid-Open No. 2009-0131399 (Cosmetic Composition Containing Mixed Extract of Natural Plant Having Anti-inflammation and Skin Stimulation Relief Effect, 2009.12.29. Korean Patent No. 508402 (herbal medicine extract having hepatitis inhibiting activity, registered on Aug. 05, 2005)

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. 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., Barrier protective effects of withaferin in HMGB1-induced inflammatory responses in both cellular and animal models. 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. Toxicol. Appl. Pharm. 2012, 262, 91-98. 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.

It is an object of the present invention to provide a composition for preventing or treating septicemia which contains a psittacosis extract or a meggestidine main glycoside isolated therefrom.

The present invention is concerned is selected from the group consisting of formulas to as dendeuro side A of the first (Rhododendroside A, compound 1), blood Cri Ono side A (Picrionoside A, Compound 2) and Ikari side B ₆ (Icariside B _6, Compound 3) ( Rhododendron brachycarpum ) extract containing at least one meglutine main glycoside. The present invention also relates to a composition for preventing or treating sepsis.

[Chemical Formula 1]

The Rhododendron phloem extract may be obtained by extractive condensation of Rhododendron with water, a lower alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof.

The Rhodiola extract has an effect of inhibiting the expression of HMGB1 (high mobility group box 1).

The invention further also dendeuro side A of Formula 1 (Rhododendroside A, compound 1), blood Cri Ono side A (Picrionoside A, Compound 2) and Ikari side B ₆ selected from the group consisting of (Icariside B _6, Compound 3) The present invention also relates to a composition for preventing or treating septicemia comprising at least one meglutine main glycoside, wherein the meglutine main glycosides also have an effect of inhibiting the expression of HMGB1 (high mobility group box 1).

In another aspect, the present invention is selected from the group consisting of dendeuro side A (Rhododendroside A, compound 1), blood Cri Ono side A (Picrionoside A, Compound 2) and Ikari side B ₆ (Icariside B _6, Compound 3) also of the general formula (1) Which contains at least one meggystine main glycoside, which is effective for preventing or ameliorating sepsis.

The present invention also provides Rhododendroside A (Compound 1) as a novel compound having the following chemical structure, and provides a method for isolating rhodendroside A from rhizome.

Hereinafter, the present invention will be described in detail.

The Rhododendron phloem extract may be obtained by extractive condensation of Rhododendron with water, a lower alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof. The extraction conditions are not limited, but the water, the lower alcohol having 1 to 4 carbon atoms, or the mixed solvent thereof is preferably added in an amount of 1 to 20 times the weight of the vinegar, 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 Rhododendron phloem extract can be obtained by a known method which is used for extractive extraction of plant components, such as extraction with water or various organic solvents, distribution of organic solvent and water, column chromatography, May be fractionated or purified and used.

Preferably, the Rhododendron phloem extract is obtained by suspending a Rhododendron phloxus extract in water by extracting and concentrating Rhododendron with water, a lower alcohol having 1 to 4 carbon atoms or a mixed solvent thereof, and then adding to the suspension, hexane, chloroform, ethyl acetate, Butanol, and the like. Preferably, the Rhizome of the Rhizome of the Rhizome is preferably a concentrate of the hexane layer obtained by suspending the Rhizome of the Rhizoma of the Rhizome by extracting and concentrating the rhizome with water, a lower alcohol having 1 to 4 carbon atoms or a mixed solvent thereof, 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) according to the present invention are megastigmane glucosides compounds isolated from the rhizome extract or Rhizoma fractions. Meanwhile, the other fractionation conditions are not limited. However, after adding a water of 1 to 50 times the weight of the Pseudomonas sp. Extract to the Pseudomonas sp. Extract to prepare a suspension, 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 fractionate the psyllium extract of the present invention or to separate the Megastich main glycoside from the silica gel column chromatography, LH-20 column chromatography (LH Ion exchange resin chromatography, medium pressure liquid chromatography (MPLC), thin layer chromatography (TLC), silica gel gel chromatography vacuum liquid chromatography, reversed phase chromatography, and high performance liquid chromatography.

The above-mentioned Pseudomonas sp. Extract can be extracted from all parts selected from outbreaks, roots, leaves, branches, stems, fruits and flowers of Pseudomonas sp.

The present invention also provides a novel compound, Rhododendroside A (Compound 1), and provides a method for isolating Rhodendroside A from Rhodiola. Preferably, Rhodendroside A is obtained by suspending a Rhododendron aquaticus extract in water, a lower alcohol having 1 to 4 carbon atoms or a mixed solvent thereof, and extracting the Rhododendron sphaeroa extract with water, and then mixing with hexane to obtain a hexane layer, A chloroform layer obtained by mixing chloroform with the residue (water layer) remaining after removal of the chloroform layer, an ethyl acetate layer obtained by mixing ethyl acetate with the residue (water layer) remaining after removing the chloroform layer, a residue (water layer) remaining after removing the ethyl acetate layer, , Or a concentrate of the butanol layer obtained by mixing butanol, or a concentrate of the residue (water layer) remaining after removing the butanol layer.

In addition, the present invention also dendeuro side A of Formula 1 (Rhododendroside A, compound 1), blood Cri Ono side A (Picrionoside A, Compound 2) and Ikari side B ₆ from the group consisting of (Icariside B _6, Compound 3) The present invention provides a pharmaceutical composition for preventing or treating septicemia containing a meganegget extract or a meggestidine main glycoside separated therefrom, which comprises at least one selected megestigin main glycoside. 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 the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

The dosage of the phytophthora extract of the present invention or the meggestidine main glycoside isolated therefrom, or the pharmaceutical composition containing the same, depends on the age, sex, body weight of the subject to be treated, the severity of the specific disease or pathological condition to be treated, , The route of administration and the judgment of the prescriber. Dosage determinations based on these factors are within the level of ordinary skill in the art and generally the dosage ranges from 0.01 mg / kg / day to approximately 2000 mg / kg / day. A more preferable dosage is 1 mg / kg / day to 500 mg / kg / day. The administration may be carried out once a day or divided into several times. The dose is not intended to limit the scope of the invention in any way.

The pharmaceutical composition 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.

In addition, the present invention also dendeuro side A of Formula 1 (Rhododendroside A, compound 1), blood Cri Ono side A (Picrionoside A, Compound 2) and Ikari side B ₆ from the group consisting of (Icariside B _6, Compound 3) The present invention provides a health functional food for preventing or ameliorating septicemia including a psittacosis extract and a pharmaceutically acceptable food-aid additive including at least one selected megestigine main glycoside. The Rhododendron phloem extract may be added in an amount of 0.001 to 100% by weight to the health functional food of the present invention. The health functional food of the present invention includes forms such as tablets, capsules, pills, and liquids. 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 septicemia, which contains a moxa agar extract or a megastigue main glycoside isolated therefrom, wherein the composition is effective for inhibiting the expression of HMGB1 (high mobility group box 1) in vascular endothelial cells Can be easily used as a health functional food for preventing or treating sepsis or for preventing or treating sepsis.

Figure 1 shows the COSY (-) and HMBC (-) correlation of Compound 1 of the present invention.
Figure 2 shows GC analysis spectral results for acid hydrolysis of Compound 1 of the present invention.
Figure 3 is a diagram showing predictable diastereomers for Compound 1 of the present invention.
Figure 4 shows immunofluorescence staining results showing that Compounds 1 to 3 of the present invention inhibit F-actin damage in HMGB1-induced HUVEC sepsis cell model.
Figure 5 is a result indicating that inhibit cell wall permeability in Rhododendron methanol extract (MeOH), chloroform fraction (CHCl _3), ethyl acetate fraction (EtOA), HUVEC sepsis cell models of butanol fraction (BuOH) is induced by HMGB1 of the invention to be.
Figure 6 shows the results of the inhibition of cell wall permeability in HMGB1-induced HUVEC sepsis cell model of Compounds 1-3 of the present invention.
Fig. 7 shows the results of inhibiting the production of HMGB1 in the HUVEC sepsis cell model in which the compounds 1-3 of the present invention were induced by LPS.
Figure 8 shows the results of the inhibition of cell wall permeability in the HUVEC sepsis cell model in which the compounds 1-3 of the present invention were induced by LPS.
FIG. 9 shows the results of inhibiting the leakage of the dye injected into the blood vessels into the abdominal cavity in the animal model of sepsis in which the compounds 1 to 3 of the present invention were induced by HMGB1.
FIG. 10 shows the results of confirming the survival rate of the sepsis animals induced by CLP surgery according to Compounds 1 to 3 of the present invention.

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

≪ Example 1 > Separation of Compound from Phenotype &

The leaves of Rhododendron were obtained from the cultivated ranch plant in Gwangju, Jeollanam - do, and the dried rhododendron leaves (25 ㎏) were extracted with methanol twice at room temperature for one week (250 ℓ each) ). The extract (3 kg) was suspended in water (10 L), and then fractionated with n -hexane (10 L) (repeated three times) and concentrated to obtain n -hexane fraction. The chloroform layer obtained by adding chloroform to the water layer (residue) separated from the n - hexane layer was concentrated to obtain a chloroform fraction. Ethyl acetate was added to the chloroform layer and the separated water layer (residue), and the ethyl acetate layer was concentrated to obtain ethyl acetate fraction. The butanol layer obtained by adding butanol to the ethyl acetate layer and the separated water layer (residue) was concentrated to obtain a butanol fraction. Each of the solvents was added in an amount of 10 L each, and repeatedly extracted three times with the same solvent. Finally, 438 g of n -hexane fraction, 140 g of chloroform fraction, 450 g of ethyl acetate fraction and 320 g of butanol fraction were obtained.

The butanol fraction (320 g) was fractionated by silica gel column VLC and elution conditions of dichloromethane: methanol: water (10: 1 -> 2: 1: 0.1) to obtain six small fractions (Fr. B1-B6). Of these, Fr. Seven fractions were obtained using elution conditions of B1 (10 g) with MPLC (C18 SNAP Cartridge KP-C ₁₈ -HS, 340 g) and methanol: water (3: 7 → 7: 3) -B17). Of these, Fr. B14 (1.46 g) using HPLC (Luna C18 (2) column : 250 × 21.20 ㎜, 20% (v / v) MeCN, flow rate: 6 ㎖ / min) to perform the compound 2 (26 ㎎, t _R 40.2 min) and compound 3 (3.8 mg, t _R 53.6 min) were obtained. Fr. B2 (22 g) was fractionated using MPLC (C18 SNAP Cartridge KP-C18-HS, 340 g) and elution conditions of acetone: methanol: water (0: 0: 100 → 12: 28: 60) (B21-B29). Of these, Fr. B26 (1.7 g) was subjected to silica gel MPLC (SNAP Cartridge KP-Sil, 120 g) and elution conditions of ethyl acetate: methanol (98: 2 -> 90: 10) to obtain 5 small fractions (B261-B265). Of these, Fr. Compound 2 (2 mg) was obtained by using silica gel column chromatography (2 x 80 cm) and elution conditions of chloroform: acetone: water (1: 2: 0.1) using B262 (92 mg).

≪ Example 2: Identification of physicochemical properties of a compound >

Example 2-1. Rhododendoside A (Compound 1)

Rhododendrosidea;

yellow amorphous powder;

: -15.4 (c 0.1, MeOH); [alpha]

: -15.4 (c 0.1, MeOH);

HR-ESI-MS: m / z 411.1996 [M + Na] + (calcd for C 19 H 32 O 8 Na, 411.1995.).

¹ H-NMR (methanol- d 4, 600 MHz) (see Table 1);

^{13 C-NMR (methanol- d 4} , 150 MHz) ( see Table 1).

In Table 1, Exp. Is the result of an analysis of Compound 1 with an NMR spectrometer, and I and III of Cal. Are predictable stereoisomers of Compound 1, CS analysis of I and III calculated using a supercomputer Value.

The compound 1 is yellow amorphous powder, and the molecular formula according to HR-ESI-MS data is C ₁₉ H ₃₂ O ₈ Na (obsd. [M + Na] ⁺ , m / z 411.1996; calcd . [M + Na] < ⁺ & gt ^; , m / z 411.1995). The ¹ H NMR spectral results of Table 1 show that Compound 1 is an olefinic proton, δ _H 4.51, anomeric proton, δ _H 4.43, oxymethine proton, δ _H 4.16, allylic, δ _H 1.66) and three tertiary methyl groups (δ _H 1.30, 1.07, 1.03). The ¹³ C NMR spectral results of Table 1 show that the glucopyranosyl moiety (δ _C 102.8, 75.1, 78.0, 71.6, 77.8, 62.7), two olefin carbons (δ _C 149.4, 97.6), oxymetin carbon _C 73.2), three quaternary carbons (δ _C 81.5, 72.0, 40.7), three methylene carbons (δ _C 44.7, 41.4 and 29.9), one allyl (δ _C 20.0) Indicating that there are 19 resonances involving six carbon resonances to which the tertiary methyl carbon (delta _C 27.4, 24.6, 22.7) belongs. These spectral data (spectroscopic data) is similar to the MB bus teak main glycosides of Ikari side _{B 2 (icariside B 2) (} Bioorg. Med. Chem. Lett. 2012, 22, 6116-6119). This means that compound 1 also has the structure of megestigin main glycoside. The difference in NMR data between icari-side B ₂ and compound 1 is the change in the quantum signal member and the acetylmethyl group of the trans -olefin group (conjugated trans- olefinic group). Compound 1 is the first cyclic megastide main structure produced by cyclization of C-6 methyl vinyl ketone of icaricide B ₂ to form an oxirane moiety, which is H-8 (隆_H 4.51) and _{C-6 (δ C 72.0)} , C-7 (δ C 29.9), C-9 (δ C 149.4), C-10 (δ C 20.0), H-7 (δ H 2.34, 1.99) and the C- 6 (? _C 72.0), C-8 (? _C 97.6) and C-9 (? _C 149.4). These hexahydro--4 H - chromen moiety generation and unique cycloalkyl Mega styryl the main scaffold (scaffold unique cyclomegastigmane) of (hexahydro-4 H -chromene moiety) has not been previously been confirmed. The substitution positions of aglycone and monosaccharide are confirmed from the HMBC correlation of H-1 '(δ _H 4.43) and C-3 (δ _C 73.2) (see FIG. 1). The coupling constant of the anomeric proton ( J = 7.8 Hz) means that glucose is linked to the β-glucosidic linkage.

In addition, according to the acid hydrolysis results using GC (gas chromatography) analysis (see FIG. 2), Compound 1 is found to have D-glucose (upper right D- Is almost similar). The acid hydrolysis method of Compound 1 was confirmed using the following method. Compound 1 was dissolved in 10% (v / v) HCl (1 ml) and heated to 85 ° C for 3 hours, and the residue was fractionated with ethanol and water. The water layer was evaporated and the residue was dissolved in anhydrous pyridine (100 μl) and 0.1 M L-cysteine methyl ester hydrochloride (100 μl) was added. After heating at 60 DEG C for 1.5 hours, the residue was dried and trimethylsilylated by adding 1-trimethylsilylimidazole solution (0.5 mL) .The solution was heated at 60 DEG C for 5 minutes behind the product obtained dried water and n -.. this was partitioned with hexane from the non-polar layer (layer n -hexane) was used for the GC analysis of monosaccharide moiety (monosaccharide moiety) is D- glucose standard product (t _R 16.21 min) and Respectively.

The association structure of aglycons is confirmed by the correlation of NOE with a pair constant. The coupling constants of H-3 (tt, 12.6, 4.1 Hz) means that there is a cyclohexane ring of the diastereomer I or diastereomer II (see FIG. 3A). The association structure of C-5 is arranged based on strong NOE correlation between H-3 (δ _H 4.16) and H-12 (δ _H 1.07), and H-3 (δ _H 4.16) and H-13 _{< / RTI > H < RTI ID = 0.0 >} 1.30) _{< /} RTI > Using the information on the dihedral constraints and spatial distance information using the MMFF94 force field (Schrodinger LLC.) With the above data for compound 1, two possible diastereoisomers a:;: (diastereomers I 3 S , 5 R, 6 R II 3 S, 5 R, 6 S) is proposed (see Fig. 3A). Between diastereomer _{I H-12 (δ H 1.07} ) and _H-8 (δ H 4.51) between it had a NOE _{correlation, H-11 (δ H 1.03} ) and _H-13 (δ H 1.30) in NOE There is no correlation. However, in diastereoisomer II this is possible. As a result of the NOE test, Compound 1 showed a NOE correlation corresponding to the diastereomer I, so that the relative stereostructure of Compound 1 was determined as a diastereomer I (see FIG. 3A).

Because of the quantitative limitation of compound 1, the absolute configuration was determined using GIAO NMR CS (gauge-invariant atomic orbital NMR chemical shift) calculations according to DP4 analysis. On the other hand, because of the D-glucopyranosyl moiety of compound 1, diastereoisomers I and III having an enantiomeric aglycone moiety of the enantiomer are considered as the structure of compound 1 ( 3B). Confirmation of the diastereoisomers I and III was performed using a Macromodel (Schrodinger LLC.) And a GIAO shield constant using the Gaussian 09 package (Gaussian Inc. B3LYP / 6-31G (d, p) level) (GIAO shielding constant calculations).

CS values were Boltzmann-averaged based on MMFF94 potential energy. The averaged values were calculated by DP4 statistical analysis.

Statistical analysis of DP4 disclosed in FIG. 3C showed that the absolute stereochemistry of Compound 1 was 98.4% consistent with the stereochemistry of I.

The absolute stereostructure of Compound 1 was once again proven by chemically predicting the pathway biosynthesized from Compounds 2 and 3. That is, the enzymatic epoxidation of compound 3 is induced via a 3? -Equatorial substituent through the following reaction scheme 1, and oxirane (compound 4) is formed. Thereafter, it can be seen that Compound 1 is formed by using the cyclization and dehydration reaction through Compound 5.

[Reaction Scheme 1]

Thus, the synthesis by the above results, the compound 1 (3 S, 5 R, 6 R) -1,1,5,9- tetramethyl -3- β -D- glucopyranosyl-oxy -2,3,4, 5-tetrahydro--7 H - chromen-6-ol ((3 S, 5 R, 6 R) -1,1,5,9-tetramethyl-3- β -D-glucopyranosyloxy-2,3,4, also has a chemical structure of the 5-tetrahydro-7 H -chromen- 6-ol) was determined by dendeuro side a (rhododendroside a).

Example 2-2. Pycryonoside A (Compound 2)

Picrionosidea;

Yellow amorphous powder;

ESI-MS m / z 371 [M + H] < ⁺ >;

[[alpha]]: + 108.8 (c0.1, MeOH);

^{1 H-NMR (250 MHz,} pyridine- d 5) δ H 6.63 (1H, dd, J = 15.8, 10.0 Hz, H-7), 6.17 (1H, d, J = 15.8 Hz, H-8), 5.91 (1H, brs, H-4), 4.98 (1H, d, J = 7.7 Hz, H-1 '), 4.50 (1H, m, H-3), 4.58-4.02 ~ H-6 '), 2.42 (1H, d, J = 9.9 Hz, H-6), 2.24 (3H, s, H-10), 1.84 (1H, d, J = 13.6, 5.9 Hz, H-2a ), 1.69 (1H, d, J = 13.6, 5.8 Hz, H-2b), 1.49 (3H, s, H- 10);

¹³ C-NMR (63 MHz, pyridine- d ₅ )? _C 197.8 (C-9), 147.5 (C-7), 135.5 (C-3 '), 78.9 (C-5'), 75.6 (C-2 '), 72.7 (C-6), 54.8 (C-6), 40.4 (C-2), 34.0 (C-1), 29.5 -13).

Examples 2-3. Ikari Side B ₆ (Compound 3)

Icariside B ₆ ;

Yellow amorphous powder;

ESI-MS m / z 373 [M + H];

[[alpha]]: 59 (c0.1, MeOH);

¹ H-NMR (600 MHz, MeOD)? _H 4.44 (1H, d, J = 7.8 Hz, H-1 '), 4.08 m, H-4 '), 3.16 (1H, m, H-5'), 3.69 M, H-2 '), 2.56 (2H, ddd, J = 9.9, 6.2, 3.4 Hz, H-8), 2.36 (1H, m, H-7b), 2.15 (3H, s, H-10), 2.05 -13), 1.52 (1H, t, J = 12.1 Hz, H-2b), 1.06 (6H, s, H-11, H-12);

¹³ C-NMR (150 MHz, MeOD)? _C 211.4 (C-9), 137.5 (C-6), 126.1 (C-5), 102.3 (C-5 '), 75.1 (C-2'), 73.1 (C-3), 71.6 ), 39.7 (C-4), 38.7 (C-1), 30.0 (C-12), 29.7 .

Example 3 Confirmation of F-actin Damage Inhibitory Effect in a Sepsis Cell Model [

Compounds 1 to 3 inhibited HMGB1 mediated vascular injury and confirmed the therapeutic effect of sepsis. Compounds 1 to 3 were treated with HUVEC (human umbilical vein endothelial cells), treated with HMGB1 for F-actin damage (a model for damage to blood vessel wall due to sepsis), and phalloidin -Actin (F-actin) was subjected to immunofluorescence staining.

HUVEC cells were obtained from Cambrex Bio Science (Charles City, IA, USA) and cultured with EBM-2 basal media and growth supplement (Cambrex Bio Science) supplied thereto. For immunofluorescence staining, HUVECs were incubated with 10% (v / v) FBS in glass cover slips coated with 0.05% (w / v) poly-L-lysine And cultured for 48 hours using a culture medium (EGM-2). To induce cell wall damage, each compound (10 μM) was pretreated with HUVEC for 6 hours and then treated with 1 μg / ml HMGB1 (Abnova) for 16 hours. After the compound treatment, cells were fixed by treatment with 4% formaldehyde in PBS (phosphate buffered saline, v / v) at room temperature for 15 min. After removing the cell fixation solution, the cells were treated with 0.05% Triton X-100 in PBS (v / v) for 15 minutes to permeabilize the cells. Then, the cells were washed with 5% BSA (bovine serum albumin in PBS, w / v) and incubated at 4 ° C overnight for more than 18 hours. (Fluorescein phalloidin, F432, Molecular Probes, Invitrogen) labeled with fluorochrome and mouse monoclonal antibody (1: 400, diluted in 5% [w / v] The reaction was carried out overnight over 18 hours and confirmed by fluorescence microscopy (Carl Zeiss, AG, Germany) at a magnification of 400 ×.

The damaged area of the cell membrane is indicated by an arrow in Fig. Referring to FIG. 4, it can be seen that the compounds 1 to 3 restored HMGB1-induced F-actin damage.

Example 4 Confirmation of the effect of inhibiting the permeability of vascular cells in a sepsis cell model [

(1 ~ 10 / / ml) and Compound 1 ~ 3 (1 ~ 10 M) were treated with HUVEC in concentrations of methanol extract, chloroform fraction, ethylacetate fraction and butanol fraction of the present invention and each compound was induced through HMGB1 (Restoration of permeability due to the minute puncture of blood vessels). For this purpose, permeability to HUVEC was confirmed, and Evans blue-bound albumin in the cell layer cultured using a 2-compartment chamber model (Blood 2011, 118, 3952-3959) Was measured quantitatively.

HUVECs were cultured for 3 days in a transwell of 12 mm diameter with 3 占 pore size at 5 占⁰⁴ / well. When cells were filled in each transwell, each compound was treated for 6 hours, then treated with HMGB1 (1 μg / ml) for 16 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 FIGS. 5 and 6. FIG.

5 and 6, the methanol extracts of Phenological Veterinary Methanol (MeOH), the chloroform fraction (CHCl ₃ ), the ethyl acetate fraction (EtOA) and the butanol fraction (BuOH) (1 to 10 μg / (10 μM = 3.88 ㎍ / ㎖) in the HMGB1-induced septicemia model, indicating that the inhibition of the permeability due to damage to the blood vessel wall is superior to that of the HMGB1-induced sepsis model (1 to 10 μM) , Compound 2 [10 μM = 3.7 μg / ml] and compound 3 [10 μM = 3.72 μg / ml] are about three times more active than the Rhizome extract or fraction [10 μg / ml].

Example 5. Confirmation of Inhibitory Effect of LPS-induced Sepsis Response < RTI ID = 0.0 >

HUVECs were cultured in 6-well plates and treated with LPS (lipopolysaccharide, 100 ng / ml) for 100 h (control treated with saline). To perform competitive enzyme-linked immunosorbent assays (ELISA) using HUVEC cell culture media, 0.02% sodium azide was added to 96-well plastic flat microtiter plates (Corning, NY, USA) 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 min) using a protein separation reagent containing RIPA lysis buffer (Upstate Biotechnology, USA) at 4 ° C for 1 hour by vortexing .

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-goat 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, all reactions were stopped with 50 μl of 8 NH ₂ SO ₄ and the absorbance was confirmed at 490 nm.

Referring to FIG. 7, it can be seen that the compounds 1 to 3 of the present invention have an excellent effect of inhibiting the production of HMGB1 in the LPS induced HUVEC septic response.

In addition, the cell permeability test was carried out by the method described in Example 4 using HUVEC treated with LPS (100 ng / ml, 100 hours) as described above, and the results are shown in FIG.

Referring to FIG. 8, it can be seen that the compounds 1 to 3 of the present invention inhibit cell permeability even after the LPS-induced sepsis reaction occurs.

Example 6 Confirmation of Vascular Cell Permeability Suppression Effect in an Animal Model of Sepsis [

Male C57BL / 6 mice were prepared (10 mice per group), 6-7 week old mice and 18-20 g body weight were purchased and adapted for laboratory use for 12 days. (W / v) Evans blue (1 mg / mouse) which had been treated with intravenous injection of Compound 1 to 3 (7.7 쨉 g / mouse) Solution and HMGB1 (2 [mu] g / mouse) were intravenously injected. After 16 hours, the mice were sacrificed and peritoneal exudate was collected by centrifugation at 200 × g for 10 minutes. The absorbance of the supernatant of the centrifugate 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.

In FIG. 9, the negative control group was treated with DMSO, which is a solvent of the compound, and the leakage amount of the dye between the negative control group and the negative control group was almost the same. In other words, in the groups not treated with HMGB1 (2 μg / mouse), dye leakage did not occur regardless of treatment with the compound. On the other hand, the positive control group treated with HMGB1 showed a large amount of dye leaching, and it was confirmed that the compounds 1 to 3 significantly inhibited the leakage of the dye due to HMGB1. Thus, it can be shown that the compounds inhibit sepsis by protecting the vascular wall damage.

Example 7. Confirmation of survival rate in animal model of sepsis < RTI ID = 0.0 >

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, a 2 cm incision was made in the abdomen to expose the cecum and the nearby intestine. The cecum at 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 to leak through the puncture site. The excreta were exposed to the abdominal cavity and the open area was sealed with a 4.0-silk suture. The sham-sham-operated mice (○) were simply stabbed and re-stitched without performing the step of tying and bursting the cecum. Sepsis was induced in the mice by the CLP procedure and each compound (7.7 [mu] g / mouse) was treated with each mouse (10 mice per group) intravenously after 12 hours and 50 hours of CLP surgery. A positive control (●) performed only with CLP was treated with a saline solution instead of the compound. Each mouse was exposed to sepsis symptoms 24 hours after CLP surgery, with symptoms such as shivering, bristled hair or weakness. Survival of the mice was confirmed from 6 hours after CLP surgery to 132 hours (observed according to Kaplan-Meier survival analysis), and the survival results of the mice over time are shown in FIG.

10, it was confirmed that the mouse group to which the compounds 1-3 were administered had a survival rate of 60% (Compound 1), 40% (Compound 2) and 40% (Compound 3) at 132 hours after CLP surgery And the treatment effect of sepsis is excellent.

<Example 8> Toxicity test>

Example 8-1. Acute toxicity

This experiment was conducted to investigate the toxicity of the compounds of the present invention (Rhodendroside A) and methanol extract of Rhododendron vinegar to an animal body in an acute manner (within 24 hours) in a short period of time and to determine the mortality rate . An ICR mouse line, a common mouse, was assigned to each group of 10 mice. In the experimental group, Compound 1 of the present invention and methanol extract of Rhododendron were mixed with the PEG-400: Tween-80: ethanol (8: 1: 1, v: v: (8: 1: 1, v: v: v). After 24 hours of administration, the mice were found to survive in the control group and in the experimental group administered with the Compound 1 of the present invention and the methanol extract of the Rhizome Vaccine at a concentration of 2 g / kg / day.

Example 8-2. Organ organs toxicity test in experimental group and control group

In the long-term toxicity test, Compound 1 (Rhodendroside A) and Rhodiola methanol extract of the present invention were administered to C57BL / 6J mice (10 mice per group) for 8 weeks at each concentration. (8: 1: 1, v: v: PEG-400: Tween-80: ethanol) was administered to the experimental group to which Compound 1 and the Rhodiola methanol extract of the present invention were administered, Blood samples were collected from the control animals at 8 weeks after the administration. The blood concentrations of glutamate-pyruvate transferase (GPT) and blood urea nitrogen (BUN) were measured using Select E (Vital Scientific NV, Netherland). As a result, GPT, which is known to be related to hepatotoxicity, and BUN, which is known to be related to renal toxicity, showed no significant difference compared to the control group. In addition, liver and kidney were cut from each animal, 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 the butanol fraction of the present invention was mixed with 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 butanol fraction of the present invention, 0.6 g of sodium chloride and 0.1 g of ascorbic acid were dissolved in distilled water to make 100 ml. This solution was placed in a bottle and sterilized by heating at 20 DEG C for 30 minutes.

<Formulation Example 2: Food Preparation>

Formulation Example 2-1. Manufacture of cooking seasonings

The butanol fraction of the present invention was added to the cooking seasoning at 1 wt% to prepare a cooking sauce for health promotion.

Formulation Example 2-2. Manufacture of flour food products

The butanol fraction of the present invention was added to wheat flour at 0.1 wt%, and bread, cake, cookies, crackers and noodles were prepared by using this mixture to prepare a food for health promotion.

Preparation Example 2-3. Manufacture of soups and gravies

The butanol fraction of the present invention was added to the soup and juice at 0.1 wt% to prepare health promotion soup and juice.

Formulation Example 2-4. Manufacture of dairy products

The butanol fraction of the present invention was added to milk in an amount of 0.1 wt%, and various dairy products such as butter and ice cream were prepared using the milk.

Formulation Example 2-5. Vegetable juice manufacturing

0.5 g of the butanol fraction of the present invention was added to 1,000 ml of tomato juice or carrot juice to prepare health promotion vegetable juice.

Formulation Example 2-6. Manufacture of fruit juice

0.1 g of the butanol fraction of the present invention was added to 1,000 ml of apple juice or grape juice to prepare fruit juice for health promotion.

Claims (10)

( Rhododendron brachycarpum ) extract comprising rhododendroside A (compound 1) of the following formula 1,
Wherein the above-mentioned Rhododendron phloem extract is a fraction of a lower alcohol extract having 1 to 4 carbon atoms in the Rhododendron sulphate or a fraction selected from the group consisting of hexane, chloroform, ethyl acetate and butanol after suspending the lower alcohol extract in water. &Lt; / RTI &gt;
[Chemical Formula 1]

delete The method according to claim 1,
The composition for preventing or treating septicemia according to any one of claims 1 to 3, wherein the extract of Phenotypes is effective for inhibiting the expression of HMGB1 (high mobility group box 1). A composition for preventing or treating septicemia, which comprises Rhododendroside A (Compound 1) of the following formula (1).
[Chemical Formula 1]

5. The method of claim 4,
Wherein Rhodendroside A has an effect of inhibiting the expression of HMGB1 (high mobility group box 1). ( Rhododendron brachycarpum ) extract comprising rhododendroside A (compound 1) of the following formula 1,
Wherein the above-mentioned Rhododendron phloem extract is a fraction of a lower alcohol extract having 1 to 4 carbon atoms in the Rhododendron sulphate or a fraction selected from the group consisting of hexane, chloroform, ethyl acetate and butanol after suspending the lower alcohol extract in water. For the prevention or amelioration of the health functional food.
[Chemical Formula 1]

delete The method according to claim 6,
Wherein the extract of Rhododendron japonica has an effect of inhibiting the expression of HMGB1 (high mobility group box 1). Rhododendroside A (Compound 1) as a novel compound having the following chemical structure.

delete

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