WO2009148287A2 - Novel α-iso-cubebene compound extracted from schisandra chinensis and composition containing the compound as active ingredient for preventing and treating inflammatory diseases - Google Patents

Abstract

The present invention relates to a novel α-iso-cubebene compound which is extracted from Schisandra chinensis. As a result of using a TNF-α-stimulated HUVEC model, the extracted novel α-iso-cubebene compound has been proven to effectively suppress the generation of ROS, the expression of VCAM-1 and E-selectin, the activation of NF-B transcription factor and monocyte adhesion to endothelial cells. Especially, while the α-iso-cubebene suppresses TNF-α-induced expression of VCAM-1 and E-selectin which perform a pivotal role in in-vitro model of early atherogenesis, it does not change the expression of ICAM-1. In addition, during early atherogenesis, a cytokine such as TNF-α activates membrane-bound NADPH oxidase and stimulates intracellular ROS generation. Then, the cytokine activates an oxidation/reduction-sensitive transcription pathway such as NF-B and induces the expression of endothelial adhesion molecules. Since the α-iso-cubebene reduces the generation of ROS and suppresses the activation of oxidation/reduction-sensitive transcription factor NF-B, it suppresses TNF-α-stimulated VCAM-1 expression and monocyte adhesion to endothelial cells. Therefore, the α-iso-cubebene provides a composition for preventing and treating inflammation.

Description

Novel α-ISO-CUBEBENE Compound Extracted from Schisandra chinensis and Composition for the Prevention and Treatment of Anti-inflammatory Disease Using the Same

The present invention relates to a novel α-iso-cubebene compound extracted from Schisandra chinensis, and more particularly to a novel compound that can be used as a composition for the prevention and treatment of anti-inflammatory diseases.

 Schisandra chinensis has been used as a traditional medicinal plant for thousands of years in Korea, China and Japan. In Chinese medicine, Schisandra chinensis is used in the lungs and kidney channels and stomachs, and is used for defects in the kidneys and lungs that cough and asthma out of breath, to stop diarrhea, to stop cold sweating, for forgetfulness and insomnia It is known to soften the mind. Recently, many studies have been conducted in the pharmacological or chemical aspects of Schizandra chinensis extract. As a result, dibenzocyclooctadiene-based lignans extracted from Schisandra chinensis are known to inhibit anti hepatitis, promote liver regeneration, and inhibit liver cancer and lipid peroxidation. Examination of this bioactive effect of Schisandra chinensis may be possible as crude extraction, but finally, it should be able to separate pure substance and increase its value. In addition, standardization and standardization by establishing a standard for evaluating the quality based on the substance when such an effect is verified can be said to be urgent.

 In particular, dibenzocyclooctadiene-based lignans and extracts extracted from Schisandra chinensis are high in medicinal value and edible, so they are mostly imported from China despite being a good material for product development. There have been many studies on Schisandra chinensis, and about 30 kinds of lignans have been identified. In Korea, only six kinds of lignans have been extracted to verify the effect of NFAT (nearly factor of activated T-cell) transcription.

 Expression of anti-inflammatory genes in the vessel wall, on the other hand, is a proven issue in the early pathogenesis leading to the development of atherosclerosis. Among these are the cytoplasmic adhesion molecules VCAM-1, E-selectin and ICAM-1. Pro-inflammatory signals, including cytokines and oxidative stress, are known to play a prominent role in the pathogenesis of coronary artery disease along many risk factors such as hypertension, hyperlipidemia, hypertension and smoking (Kunsch et al., 2004).

 In particular, reactive oxygen species (ROS) serve as common intracellular messengers for a number of redox-sensitive pathways leading to vascular endothelial adhesion molecule expression (Griendling et al., 2000; Harrison et al., 2003; Ross, 1999). Factors with antioxidant activity such as probecall, brain health extract and carvedilol can remove intracellular ROS when accumulated in vitro and in vivo and inhibit endothelial adhesion to monocytes by reducing the expression of multiple adhesion molecules (Chen et al., 2003a, 2004; Fruebis et al., 1997). From these results, VCAM-1 can play a crucial role in the in vitro model of early atheroogenesis. This is consistent with recent findings related to atheromatosis, in which VCAM-1 is crucial for monocyte repair for endothelial cells, the key to the development of atherosclerotic loss (Cybulsky and Gimbrone, 1991).

   During early atherosclerosis, cytokines such as TNF-α can activate membrane-bound NADPH oxidase to stimulate intracellular ROS production. They then activate redox-sensitive transcription pathways such as NF-κB to induce the expression of endothelial adhesion molecules (Kunsch and Medford, 1999; Manna et al., 1998; Martin et al., 1997; Muller et. al., 1997; Ross, 1995). Specifically, NF-κB is critical for the expression of TNF-α stimulated VCAM-1 (Chen et al., 2003b, 2004; Cominacini et al., 1997; Martin et al., 1997; Marui et al., 1993 Weber et al., 1994; Zhu et al., 1998). Pyrrolidone dithiocarbamate, an inhibitor of the NF-κB activator, strongly inhibits TNF-α-induced expression of VCAM-1 in HUVECs but does not inhibit the expression of ICAM-1. In addition, antioxidants such as probecall, brain health extract, and carvedilol down-regulate NF-κB activity to inhibit endothelial VCAM-1 expression in vitro to prevent in vivo atherosclerosis (Bridges et al., 1991; Chen; et al., 2003a, 2004; Fruebis et al., 1997; Kunsch and Medford, 1999; Marui et al., 1993; Offermann and Medford, 1994; Tardif et al., 2002; Weber et al., 1994; Zapolska- Downar et al., 2001).

The present inventors, while studying the pure separation and assay of the material extracted from Schizandra chinensis, the new cubebene sesquiteroene compound α-iso-cubebene expresses endothelial cell adhesion molecules, TNF-α stimulated human umbilical vein endothelial cells The adhesion of monocytes to endothelial cells was confirmed, thereby demonstrating the anti-inflammatory properties of α-iso-cubebene and reaching the present invention.

One feature of the present invention is to provide an α-iso-cubebene compound having the following Chemical Formula 1 extracted from Schisandra chinensis , Chemical Fomula C ₁₅ H ₂₄ , molecular weight 204.35.

[Revision 25.06.2009 under Rule 26]

One feature of the present invention is to provide a composition for the prevention and treatment of anti-inflammatory diseases containing the compound as an active ingredient.

 In the present invention, the anti-inflammatory disease is atherosclerosis, diabetes, arthritis, obesity, inflammatory bowel disease (IBD), Alzheimer's disease, multiple sclerosis, tuberculosis, sarcoidosis, hepatitis , Cholecystitis, fungal infections, gastric ulcers, asthma, atopic dermatitis, tendonitis, or nephritis.

 One feature of the present invention is a method for expressing anti-inflammatory properties by inhibiting the expression of the cell surface adhesion molecule VCAM-1 increased by TNF-α in vascular endothelial cells by administering to a patient in need of the above-described prophylactic and therapeutic compositions. To provide.

 One feature of the present invention is a method of expressing anti-inflammatory properties by inhibiting the expression of cell surface adhesion molecule E-selectin increased by TNF-α in vascular endothelial cells by administering to a patient in need of the above-described prophylactic and therapeutic compositions. To provide.

 One feature of the present invention is a method for expressing anti-inflammatory properties by inhibiting the expression of pro-inflammatory cytokine MCP-1 increased by TNF-α in vascular endothelial cells by administering to a patient in need of the above-described prophylactic and therapeutic compositions. To provide.

 One feature of the present invention is a method of expressing anti-inflammatory properties by inhibiting the expression of pro-inflammatory cytokine IL-6 increased by TNF-α in vascular endothelial cells by administering to a patient in need of the aforementioned prophylactic and therapeutic compositions. To provide.

 One feature of the present invention is a method of expressing anti-inflammatory properties by inhibiting the expression of pro-inflammatory cytokine IL-8 increased by TNF-α in vascular endothelial cells by administering to a patient in need of the prophylactic and therapeutic compositions described above. To provide.

 One feature of the present invention is to provide a method for expressing anti-inflammatory properties by inhibiting adhesion between vascular endothelial cells and leukocytes by administering to the patient in need of the above-described prophylactic and therapeutic compositions.

 One feature of the present invention is to provide a method for expressing anti-inflammatory properties by controlling inflammation through the activity of vascular endothelial cells by administering to a patient in need of the above-described prophylactic and therapeutic compositions.

One feature of the present invention is the step of obtaining a hexane extract using the hexane from Schizandra chinensis; Adding the hexane extract to the surface of the column filled with silica gel, and sequentially adding hexane, hexane-ethylacetic acid and chloroform-methanol mixed solution to the column to obtain a plurality of fractions; Adding a first fraction of the fractions to a column packed with silica gel to obtain a subsequent fraction while flowing a CH ₂ Cl ₂ solution; And adding the obtained subsequent fraction to a column filled with silica gel to separate the pure water while flowing hexane solution to identify α-iso-cubebene compound having Chemical Fomula C ₁₅ H ₂₄ , molecular weight 204.35; It is to provide a method for extracting α-iso-cubebene compound from Schizandra consisting of.

 EMBODIMENT OF THE INVENTION Hereinafter, this invention is demonstrated concretely.

First, a novel α-iso-cubebene compound is extracted from Schisandra chinensis. At this time, the extraction method is not limited thereto, but 2 kg of Schizandra chinensis powder was obtained using hexane to obtain 308 g of hexane extract, and 120 g of the hexane extract was added to the surface of the column filled with 1 kg of silica gel, and , Hexane-ethylacet and chloroform-methanol mixed solution 15L, 16L, 58L and 19L, respectively, were sequentially added to obtain 38 fractions, of which 3.7 g of the first fraction was added to a column packed with 10 ⁵ x 3 cm silica gel. ₂ L of CH ₂ Cl ₂ solution was flowed to obtain 999 mg of the fraction. 999 mg of the fraction was added to a column filled with silica gel to flow 1 L of hexane solution to obtain 316 mg of pure material. In order to clarify the structure of the purely separated material, it is sufficient to sequentially perform a process of identifying the structure of Chemical Formula 1 having Chemical Fomula C ₁₅ H ₂₄ and a molecular weight of 204.35 using 1D and 2D NMR spectra.

[Formula 1]

[Revision 25.06.2009 under Rule 26]

 The α-iso-cubebene compound of Chemical Formula 1 was confirmed to have a prophylactic and therapeutic effect against anti-inflammatory as shown in the following examples. Specifically, as a result of using the TNF-α-stimulated HUVEC model, α-iso-cubebene effectively prevented the formation of ROS, expression of VCAM-1 and E-selectin, activation of NF-κB transcription factor and adhesion of monocytes to endothelial cells. It was confirmed that it was effectively suppressed. In particular, α-iso-cubebene inhibits TNF-α induced expression of VCAM-1 and E-selectin, which may play a decisive role in the in vitro model of early atheroogenesis, whereas expression of ICAM-1 Did not change.

   During early atherosclerosis, cytokines such as TNF-α can activate membrane-bound NADPH oxidase to stimulate intracellular ROS production. They then activate redox-sensitive transcription pathways such as NF-κB to induce the expression of endothelial adhesion molecules, thereby reducing ROS production and inhibiting the activity of the redox-sensitive transcription factor NF-κB. cubebene inhibits the expression of TNF-α-stimulated VCAM-1 and inhibits endothelial adhesion to monocytes.

 Compounds disclosed herein may be used in topical or parenteral administration, specifically in the form of a solution or liquid suspension; For oral administration, specifically in the form of tablets or capsules; Or for intranasal administration, specifically in the form of powders, gels, oily solutions, nasal drops, aerosols or mists; It can be formulated as. Formulations for parenteral administration may include conventional excipients sterile water or sterile saline, polyalkylene glycols such as polyethylene glycol, vegetable oils, hydrogenated naphthalenes, and the like. Controlled release of the compounds of the present invention can be obtained with the use of biocompatible, biodegradable lactide polymers and some copolymers of lactide / glycolide or polyoxyethylene / polyoxypropylene.

 Additional enteral delivery systems include ethylene-vinyl acetate, acetate copolymer particulates, osmotic pumps, subcutaneous insertion systems, liposomes, and the like. Formulations for inhalation administration include lactose, polyoxyethylene-9-laurylether, glycocholate or deoxycholate. Formulations for oral administration include glycocholate; Formulations for vaginal administration may include citric acid.

 The concentration of the compound in the pharmaceutically acceptable composition may depend on the dosage of the compound to be administered, the pharmacokinetic properties of the compound used and the route of administration, but in general, the compounds of the present invention are compounds 1-10 may be provided in an aqueous physiological buffer comprising% w / v. Typical dosage ranges may be administered in 1-4 fractions once at a weight of 1-100 mg / kg, preferably 2-10 mg / kg. Each fractional administration may contain the same or different compounds of the invention. The dosage can vary depending on a number of factors, including the overall health and combination of the patient and the route of administration of the selected compound.

In addition, the novel compounds of the present invention may be combined with pharmaceutically acceptable carriers, and may also be used in combination with known therapeutic agents.

Α-iso-cubebene compound isolated from the Schizandra chinensis of the present invention was not only purely isolated from highly active natural substances, it was confirmed that it has an excellent effect on anti-inflammatory diseases.

 1 is a main HMBC association diagram of new material isolated from Schizandra chinensis.

 2 is a diagram illustrating the effect of α-iso-cubebene on HUVEC cell viability, (A) the shape of HUVEC observed under a light microscope of 200x magnification, (B) HUVEC measured by the WST-1 assay The viability of each is shown.

 Figure 3 shows the effect of α-iso-cubebene on the expression of adhesion molecules in TNF-α-activated HUVECs by RT-PCR analysis, where (A) shows TNF-α treatment (20 ng / ml) is a view showing the expression level of VCAM-1, E-selectin and ICAM-1, (B) is the ratio of VCAM-1, (C) is the ratio of E-selectin, and (D) is ICAM-1 Is a graph showing the results obtained by measuring the ratio of N with a scanning density meter.

 Figure 4 shows the effect of the α-iso-cubebene on the expression of adhesion molecules in TNF-α-activated HUVEC by flow cytometry, TNF-α treatment is VCAM-1 (A), E-selectin (B ) And the effect on the expression of ICAM-1 (C).

 FIG. 5 shows the effect of α-iso-cubebene on the expression of adhesion molecules in TNF-α-activated HUVECT under immunofluorescence microscopy. TNF-α treatment was performed by VCAM-1 (A), E-selectin (B ) And the effects on the expression of ICAM-1 (C), respectively.

 FIG. 6 shows the results of quantitative adhesion assay of U937 on HUVEC, (A) is a result of observing with a light microscope of 200x magnification, and (B) is a diagram showing the result of counting the number of U937 attached.

 FIG. 7 illustrates the effect of α-iso-cubebene on TNF-α induced ROS generation in HUVECs.

 FIG. 8 shows the results of examining the effects of α-iso-cubebene on nuclear displacement in NF-κB in TNF-α-activated HUVECs by immunofluorescence microscopy (A) and Western blot analysis (B).

 9 is a diagram illustrating the effect of α-iso-cubebene on the binding activity of NF-κB having DNA in TNF-α-activated HUVEC by electrophoresis gel transfer assay, (A) is α-iso-cubebene (25 μg / ml) for 6 hours and TNF-α treatment (20 ng / ml) for 30 and 60 minutes, and then the cells are fractionated into cytoplasm and nuclear extract, (B) shows (A) Shows the result of calculating the mean ± standard deviation.

material

  The material used to extract bioactive substances from Schisandra chinensis was purchased in January 2007 from the Dongro Agricultural Cooperatives in Mungyeong.

Example 1: Extraction of substances from Schizandra chinensis

The dried Schisandra chinensis fruits collected at Mungyeong were completely ground with a grinder. 2 kg of crushed fruits were placed in a 5 L Erlenmeyer flask, filled with 3 L of hexane, sonicated for 2 hours, and the supernatant was washed with Whatman no. It filtered with 2 filter paper. Hexane extract was repeatedly extracted three times by the above method to obtain 308 g of the hexane layer. Extracted with hexane and repeatedly extracted three times with 3L of CHCl ₃ to the remaining residue to give a 14 g of chloroform layer. Extraction with chloroform and the remaining residues were extracted three times with 3 L of MeOH to obtain 1,368 g of MeOH. The extracted substances were classified into KH, KC and KM, respectively, and the neutrophil cells were measured for their activity to increase the release of intracellular calcium ions.

Example 2: Pure Separation of Material from Hexane

The material was purely separated from the hexane extract with high activity. A 100 × 10 cm column was filled with 1 kg of silica gel dissolved in hexane. 120 g of hexane extract (KH) was added to the packed column upper layer, and then 38 fractions were obtained by sequentially using a solvent of 100% hexane, hexane: EtOAC and CHCl ₃ : MeOH.

   38 fractions were selected and sent to the physiological activity verification team for a certain amount of activity for activity verification, and pure material was sequentially separated from the highly active fractions.

Example 3: Pure separation and structure identification of bioactive new substance by active fraction verification

In order to elucidate the structure of the purely separated material, the structure was identified using 1D and 2D NMR spectra, and the results were confirmed as shown in Table 1 and Formula 1. For reference, Table 1 summarizes the two-dimensional NMR correlation of the novel compounds. The structure identified by pure separation by the active fraction verification of this study proved to be a new substance.

[Revision 25.06.2009 under Rule 26]

Specifically optical rotation was recorded on a JASCO DIP-370 digital polarimeter. The IR spectra were recorded on an AATI Mattson Genesis Series FTIR. NMR spectra ( ¹ H, ¹³ C) were recorded in CDCl ₃ on a Varian Inova spectrometer at 500 MHz for protons and 125 MHz for carbon-13 using gradients and using residual solvent peaks as embedded criteria. High resolution mass spectra were recorded on a Bruker BioApex FT mass spectrometer. α-iso-cubebene was obtained as a white oil with [α] _D ²⁵ -13 ( c 0.51, CHCl ₃ ). Positive high-resolution time-of-flight mass spectrometry (HRTOFMS) showed molecular ions at m / z 222 corresponding to [M + NH ₄ ] ⁺ , thus the molecular formula was C ₁₅ H ₂₄ NH ₄ . Analysis of 1D and 2D NMR data with homo- and heteronuclear oriented and long range correlations allowed the assignment of 1H and 13C NMR resonances as shown in FIG. 1 and Table 1. ¹³ CNMR and DEPT spectra showed 15 signals comprising two olefin carbons (δ 142.5 and 117.1) and four methyl groups (δ 23.3, 19.9, 19.3 and 19.1). ¹³ CNMR / DEPT techniques also include four primary (δ 23.3, 19.9, 19.3, 19.1), three sencondary (δ 36.7, 30.9, 22.2), six tertiary (δ 117.1, 43.2, 44.7, 45.5, 48.3, 32.5), And two quaternary carbons (δ 142.5, 39.4). 1H NMR results of α-iso-cubebene shifted to 3 downlinks in 3 methyl doublet and δ = 1.68 (H-15) in the region δ = 0.790.89 (H-12, H-13, H-14) Showed a methyl signal. The absorbance of one olefin proton was detected at δ = 5.28 (H-4), while the two cyclopropane protons resonated at δ = 2.03 (H-6) and 1.67 (H-5).

 As a result of HMBC (multiple bond CH correlation), furthermore, carbon H-11 is bound to both C-6 and C-8, carbon H-7 is bound to C-1, C-5 and C-9, and H- 12 and H-13 further demonstrated binding to C-7 and C-11. The HMBC correlation result observed between H-15 and C-4, C-3 and C-2 suggests the presence of methyl groups at C-3 of the cyclopropane ring. Cross peaks were also observed between H-15 and C-2 and between C-15 and H-2 and H-4. Since H-9 was bound to methane carbon C-10 and methyl doublet H-14, a 6 membered intracyclic linkage was established. Adjacent quaternary carbon C-1 was found to correlate with H-2, H-5, H-6, H-7, H-9, H-10 and H-14, which results in a conformity with the cubebene sesquiterpene skeleton. . This structural determination of the present material was confirmed as HMBC, and the results are summarized in FIG.

As a result, when the 1D or 2D NMR is synthesized, the structure of the purely extracted material in this experiment is the same as that of Chemical Formula 1, and is Chemical Formula: C ₁₅ H ₂₄ and molecular weight: 204.35.

[Formula 1]

[Revision 25.06.2009 under Rule 26]

Cell culture system

Human umbilical vein endothelial cells (HUVEC) were obtained from Lonza (Walkersville, MD, USA). The HUVECs were converted to vascular endothelial growth factor (VEGF), fibroblast growth factor (bFGF), insulin-like growth factor-1 (IGF-1), epithelial growth factor (EGF), ascorbic acid, hydrocortisone, and 2% fetal calf serum. Cultured in EGM-2 basic medium (Lonza) containing (FBS). For this study, the HUVEC with 2-10 passages was used and cells were grown at 37 ° C. in a 5% CO ₂ humidified atmosphere. Fresh medium was changed every 3 days. Human mononuclear cell line U937 (Korean Cell Line Bank, Seoul, Korea) is contained in RPMI-1640 (Hyclone, Logan, UT, USA) containing 10% FBS (Hyclone), 100 IU / ml penicillin and 100 mg / ml streptomycin. Growing at 37 ° C. in 95% air and 5% CO ₂ atmosphere in suspension culture.

Cytotoxicity assay

 HUVEC (3x10³Cells / well) were incubated for 16 h on 96-well plates containing 0.1 ml culture medium and then treated for 6 h with or without α-iso-cubebene (10, 25 or 50 μg / ml) And then stimulated with TNF-α (20 ng / ml) for 24 h. cell Viability was measured by morphology analysis and colorimetric WST-1 transformation assay (Roche, Mannheim, Germany). 10 μl of WST-1 reagent (Roche) is added to each well, and the cells are then removed in 5% CO in a humidified incubator.₂Incubated at 37 ° C. for 2 h. Absorbance was measured at 450 nm with a microplate reader (Tecan, Mannedorf, Switzerland) and% toxicity calculated.

 The test results for the cytotoxicity of α-iso-cubebene are shown in FIG. 2. As shown in FIG. 2A, α-Iso-cubebene did not compromise the viability of HUVECs as measured by HUVEC morphology in both unstimulated and TNF-α-stimulated HUVECs. Moreover, the cytotoxicity of α-iso-cubebene was measured using the WST-1 assay (Roche). As a result, it was confirmed that α-iso-cubebene did not affect HUVEC cell viability up to a dose of up to 25 μg / ml in both unstimulated and TNF-α-stimulated HUVECs (see FIG. 2B).

Example 3: Anti-inflammatory and Treatment Effects

    Endothelial cells are structures that border the inside and outside of blood vessels of various organs and have important physiological functions that determine coagulation factor production, inflammatory cell migration, cytokine production, and blood vessel tension. Vascular endothelial cells are cell adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1), E-selectin and intercellular cell adhesion molecule-1 (IMC-1) when exposed to inflammatory stimuli such as IL-1 or TNF-α. By attaching the inflammatory cells through the plays a pivotal role in the inflammatory response. Unlike ICAM-1, which is expressed in normal vascular endothelial cells, VCAM-1 and E-selectin are not expressed in normal vascular endothelial cells, but in vascular endothelial cells by various inflammatory cytokines during inflammatory reactions. Expression is significantly induced to mediate the inflammatory response. In particular, VCAM-1 is one of the most important mediators of inflammatory response. Inflammatory responses between inflammatory and vascular cells following endothelial cell damage are important pathophysiology of early atherosclerosis. When vascular endothelial cells damaged by triggers such as hypertension, hyperglycemia, hypoxia, oxygen radicals and cholesterol do not function as a barrier of blood vessels, lipids and leukocytes in the blood penetrate into the endothelial tissue. Cell adhesion molecules are mainly responsible for the attachment of leukocytes to vascular endothelial cells, causing atherosclerotic vascular damage, and are known to be involved in the generation of atherosclerotic plaques. The interaction between leukocytes and endothelial cells by these cell adhesion molecules is the most important molecular mechanism of all inflammatory reactions. In addition to atherosclerosis, diabetes, arthritis, obesity, inflammatory bowel disease (IBD) It plays an important role in the development of various inflammatory diseases such as Alzheimer's disease, multiple sclerosis, tuberculosis, sarcoidosis, hepatitis, cholecystitis, fungal infections, gastric ulcer, asthma, atopic dermatitis, tendonitis and nephritis.

 In the normal state, VCAM-1 and E-selectin (endothelial selectin), which are not expressed in vascular endothelial cells, are induced by inflammatory cytokines IL-1β and TNF-α or bacterial lipopolysaccharide LPS (lipopolysaccharide). Therefore, the researchers were able to demonstrate the anti-inflammatory effect of the new compound through the following experiment.

Example 3-1. Flow Cytometry

 For flow cytometry, cells were first cultured in 2.4G2 (anti-Fc receptor) medium supernatants to block nonspecific antibody binding and then stained for binding with fluorescent chromium-conjugated monoclonal antibody (mAbs). Phycoerythrin (PE) -anti-ICAM-1 (CD54, HA58) and PE-anti-VCAM-1 (CD106, 51-10C9) were purchased from BD Biosciences (San Jose, CA, USA). PE-anti-E-selectin (CD62E, HCD62E) was purchased from BioLegend (San Diego, CA, USA). Background fluorescence was measured on cells stained with fluorochrome-labeled isoform-matched non-reactive mABs. The electron gate was set on HUVEC using forward and side diffusion directivity. For all experiments, cells were gated on the front and side diffusers to remove lethal cells and debris.

Reduction of VCAM-1 and E-selectin Gene Expression by α-iso-cubebene

To analyze gene expression of cell adhesion molecules in HUVECs, mRNA expression of VCAM-1, E-selectin and ICAM-1 was examined by RT-PCR analysis. Treatment of HUVECs with TNF-α (20 ng / ml) for 6, 12 and 24 hours markedly increased mRNA content for VCAM-1, E-selectin and ICAM-1 (see Figures 3A-D). Pretreatment with α-iso-cubebene (25 μg / ml) significantly reduced TNF-α-induced mRNA expression of VCAM-1 and E-selectin (from 6 h to 28% and 12 h for VCAM-1). 43% and 44% at 24 hours, and 37% at 6 hours, 50% at 12 hours and 51% at 24 hours for E-selectin; P <0.001). However, ICAM-1 is not affected by mRNA expression. (See Figures 3A-D).

Inhibition of Cell-Surface Expression of VCAM-1 and E-selectin by α-iso-cubebene

 In order to analyze the expression of cell adhesion molecules on the surface of HUVECs, expression of ICAM-1, VCAM-1 and E-selectin was evaluated by flow cytometry. HUVEC treatment with TNF-α (20 ng / ml) for 12 hours induced cell surface expression of VCAM-1, E-selectin and ICAM-1 (see Figures 4A-C). α-Iso-cubebene (25 μg / ml) significantly reduced TNF-α-induced cell surface expression of VCAM-1 and E-selectin (43.8% and 29.6% inhibition, respectively), but the expression of ICAM-1 did not affect It was not given (see Figures 4A-C).

Reduction of VCAM-1 Protein Expression by α-iso-cubebene

 Incubating HUVECs with TNF-α (20 ng / ml) for 6 hours significantly increased total protein content for VCAM-1, E-selectin and ICAM-1, as shown by immunofluorescence microscopy. (See FIGS. 5A-C). α-iso-cubebene (25 μg / ml) significantly reduced TNF-α-induced total protein expression of VCAM-1 and E-selectin (see FIGS. 5A and 5B). Expression of ICAM-1 protein was not altered by α-iso-cubebene (see FIG. 5C).

Example 3-2: Immunofluorescence Staining

 For immunofluorescence staining analysis, HUVECs were incubated on a 12 mm glass cover slip (Paul Marienfeld, Lauda-Konigshofen, Germany) coated with 50 μg / ml poly-L-lysine (Sigma, St. Louis, USA) and 0.1 Fix in cold 4% paraformaldehyde in M phosphate buffer (PB) for 10 minutes. The fixative was then removed by washing three times for 5 minutes with cold phosphate buffer solution (PBS) and the cells were then incubated with 0.5% Triton X-100 in PBS for 5 minutes. The cells were washed again with cold PBS and then incubated with 2% BSA (Sigma) at room temperature for 60 minutes.

 Discard the excess solution and cells were purified at 4 ° C. with purified anti-mouse-ICAM-1 antibody diluted 1: 100 (116102, BioLegend) or purified anti-VCAM-1 antibody diluted 1: 100 (165708, BioLegend). Incubated for -18 hours. PE-anti-E-selectin (CD62E, HCD62E) was purchased from BioLegend.

After incubation with primary antibody, the cells were washed three times with cold PBS for 5 min and diluted 1: 100 (705-096-147, Jackson ImmunoResearch Laboratories, West Grove, PA, USA) diluted affinity-purified donkey anti- The F (ab ') ₂ fractions of rat FITC-conjugated antibodies were incubated. Cells were then rinsed with cold PBS and mounted on glass slides using Vectashield (Vector Laboratories, Burlingame, Calif., USA).

 Controls for the staining procedure include the following steps: (1) the primary antibody was omitted from the staining sequence and non-immune donkey serum was used instead, and (2) the secondary antibody was omitted from the staining sequence. Labeled cells were examined with an Olympus BX50 microscope equipped with a fluorescence reflector-imager. Micrographs were acquired electronically with an Olympus DP70 digital camera at 1,360 x 1,024 pixel resolution.

Example 3-3 Cytoplasm Preparation and Nuclear Cell Extraction

After treatment as described above, HUVEC (1 × 10 ⁶ cells) was washed with cold PBS and 70 μl of Buffer A [10 mM HEPES (pH7.9), 1.5 mM MgCl ₂ , 10 mM KCl, 0.5 mM DTT, 0.5 mM PMSF and Protease inhibitor Cocktail (Sigma)] was resuspended and cultured on ice. After 15 minutes, 0.5% Nonident P (NP) -40 was added to lyse the cells and vortex for 10 seconds. It was then centrifuged at 4 ℃ for 60 seconds at 6,500 rpm to obtain a cytoplasmic cell extract. Nuclei were resuspended in 50 μl of buffer C [20 mM HEPES (pH7.9), 1.5 mM MgCl ₂ , 420 mM NaCl, 0.2 mM EDTA, 25% v / v glycerol, 0.5 mM PMSF and protease inhibitor Cocktail] Incubate on ice for 20 minutes with gentle pipetting up. Nuclear cell extracts were recovered by centrifugation at 12,000 rpm for 10 minutes at 4 ° C. Protein concentration was measured using Bradford protein assay reagent (Bio-Rad, Hercules, CA, USA).

α-Iso-cubebene does not affect the displacement of NF-κB and phosphorylation of IκBα induced by TNF-α in HUVECs.

 In order to observe whether the activity of NF-κB by TNF-α can be inhibited by α-iso-cubebene, the main component of the NF-κB complex was measured. As analyzed by immunofluorescence microscopy, TNF-α induced NF-κB (p65), which displaces the cytoplasm into the nucleus, was not inhibited by α-iso-cubebene (see FIG. 8A). The immunofluorescence microscopy analysis was confirmed by Western blot (see Figure 8B). Treatment of HUVECs with TNF-α (20 ng / ml) for 30 minutes and 60 minutes induced cytoplasmic p65 and p50 displacement into the nucleus (see FIGS. 8A and B). From these results, it was confirmed that α-Iso-cubebene treatment did not replace the displacement of p65 or p50 into the nucleus of the cytoplasm.

 In addition, inhibition of phosphorylation of IκBα and NF-κB activity by TNF-α was measured and summarized in FIG. 8B. As shown in FIG. 8B, α-iso-cubebene did not affect the phosphorylation of IκBα by TNF-α. These results demonstrate that α-iso-cubebene does not inhibit the displacement of NF-κB into the nucleus of the cytoplasm in HUVECs.

Example 3-4 Western Blot Analysis

 Equal amounts of protein samples were heated for 10 minutes at 95 ° C in sample buffer and 10% sodium dodecyl sulfate (SDS) -polyacrylamide gel electrophoresis (PAGE) using Mini-Protean II system (Bio-Rad) Separated by. Proteins were transferred onto PVDF membrane (Bio-Rad) by semi-dry transfer (Bio-Rad). The membrane was anti-NF-kB p65 (sc-109, Santa diluted 1: 1000 in 2% skim milk containing Tris-buffer saline (TBS, 20 mM Tris-HCl, 150 mM NaCl, pH 7.4) overnight at 4 ° C. Cruz Biotechnology, Santa Cruz, CA, USA), anti-NF-kB p50 (sc-7178, Santa Cruz Biotechnology) or anti-IkB-α (sc-203, Santa Cruz Biotechnology).

 After washing three times with TBS-T (TBS containing 0.1% Tween 20), the membrane was washed with a secondary antibody (goat anti-rabbit IgG HRP conjugate, sc-2004, Santa Cruz Biotechnology) diluted 1: 1000 at room temperature for 2 hours. After incubation, washed three times with TBS-T again. Immune activity was detected with an improved chemiluminator (ECL, SuperSignal West Pico Chemiluminescent Substrate kit, Pierce, Rockford, IL, USA) according to the manufacturer's instructions. Images were obtained and quantified using the LAS-3000 imaging system (Fujifilm, Tokyo, Japan).

Example 3-5: Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis

According to the manufacturer's protocol for total RNA using TRIzol reagent ^ⓡ (Invitogen, Carlsbad, CA, USA ) were isolated from each sample. Briefly, samples were transferred to tubes containing 1 ml of RNA extract. Homogenate was then extracted with chloroform, isopropanol precipitated, ethanol washed and resuspended in 30 μl of distilled water. RNA concentration and purity were measured at absorbances 260 and 280 nm. The sample exhibited an absorbance ratio of at least 1.7 (260/280). First strand cDNA was obtained by reverse transcription (RT) using 2 μg of HUVEC RNA. The reaction consisted of 0.5 μg Oligo (dT) 1218 primer (Promega, Madison, WI, USA), 50 mM TrisHCl, pH 8.3, 75 mM KCl, 3 mM MgCl ₂ , 40 mM DTT, 0.5 mM deoxynucleotide triphosphate ( dNTP) mixture (Promega), 10 units RNase inhibitor (Promega) and 200 units of MMLV reverse transcriptase (Promega) containing 25 μl buffer.

 After incubation at 42 ° C. for 60 minutes, the reaction was stopped by heating at 70 ° C. for 15 minutes. The cDNA was used as a template for amplification when using gene specific primers for human VCAM-1, E-selectin and ICAM-1PCR. Specific primers were designed for each gene (Bioneer, Daejeon, Korea) and primers for human VCAM-1 were forward primers (5 ′) corresponding to nucleotides 1780-1799 in the human VCAM-1 gene sequence (GenBank accession no. NM_001078). -GAGCTACAGCCTCTTTCTGA-3 '), and reverse primers (5'-GAGGATGCAAAATAGAGCAC-3') complementary to nucleotides 2221-2240, wherein the primers for human E-selectin are human E-selectin gene sequence (GenBank accession no. NM_000450 ) A forward primer (5′-ACCTCCACGGAAGCTATGAC-3 ′) corresponding to nucleotides 192-21 and a reverse primer (5′-TCCCAGATGAGGTACACTGA-3 ′) complementary to nucleotides 968-987 and primers for human ICAM-1 Is a forward primer (5′-CTGCAGACAGTGACCATCTA-3 ′) corresponding to nucleotides 961-980 in the human ICAM-1 gene sequence (GenBank accession no. NM000201), and nucleotides 1348-1367 Comprises a complementary reverse primer (5'-AAAGTGCCATCCTTTAGACA-3 '). They amplify 461 bp, 796 bp and 407 bp fractions and are used to analyze human VCAM-1, E-selectin and ICAM-1 transcript, respectively.

PCR amplification of cDNA was performed with cDNA samples (1 μl for VCAM-1, E-selectin and ICAM-1; 0.5 μl for GAPDH), 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 1.5 mM MgCl ₂ , 0.1% In an automated DNA amplification device (TECHNE, Teddington, UK) in a total volume of 25 μl containing TritonX-100, 0.2 mM dNTP mixture (Promega), 0.4 pmol of each primer and 5 units of Taq DNA polymerase (Promega) Was performed in. The amplification procedure was initially denatured at 94 ° C. for 5 minutes followed by denaturation at 94 ° C. for 30 seconds, primer binding to VCAM-1 and ICAM-1 at 54 ° C. for 30 seconds, primer binding to E-selectin at 55 ° C., and Primer bound to GAPDH at 50 ° C., expanded at 72 ° C. for 30 seconds, finally expanded at 72 ° C. for 10 minutes, and finished with a 4 ° C. support cycle. The number of PCR cycles was 30 for VCAM-1, E-selectin, and ICAM-1 and 25 for GAPDH.

 After PCR, the amplified product was electrophoresed in 1.5% agar gel and visualized by ethidium bromide staining under UV light emission. Band intensities of PCR products were measured using an image analysis program (MetaMorph, Universal Imaging Corporation, Downingtown, PA, USA). The data is based on VCAM-1, E-selectin and ICAM normalized with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA amplified from the same cDNA sample to correct any errors in spectrospectral RNA quantification or pipetting. Expressed as ratio of −1 mRNA.

Example 3-6 Monocyte-Endothelial Cell Attachment Assay

To investigate the effect of α-iso-cubebene on endothelial monocyte binding, the degree of adhesion to T937-activated HUVECs of U937 cells and human mononuclear cells was investigated under static conditions. HUVECs were grown under confluence in 35 mm plates, pretreated with α-iso-cubebene (25 μg / ml) for 6 hours and then stimulated with TNF-α (20 ng / ml) for 12 hours. Briefly, cells were washed twice with PBS and co-cultured with 1 × 10 ⁶ cells / well of U937 cells for 3 hours. The dish was washed by washing, unattached U937 cells were removed and washed gently with PBS twice. The cells were photographed with an Olympus BX50 microscope. Micrographs were obtained electronically at 1,360 x 1,024 pixel resolution with an Olympus DP70 digital camera. The experiment was repeated at least three times. In addition, adherent U937 cells were collected by gentle pipetting and counted using trypan blue staining using a hemocytometer.

Inhibition of monocyte adhesion to TNF-α-stimulated HUVECs by α-iso-cubebene

 The results indicate that culture of HUVEC with α-iso-cubebene significantly weakens the level of VCAM-1 (Inoue et al., 2006; Nizamutdinova et al., 2008), which is known to play an important role in attaching monocytes to EC. Seemed to let. The effect of α-iso-cubebene on the adhesion of monocytes to HUVEC after stimulation with TNF-α was investigated. That is, the number of U937 binding to HUVEC under different conditions was evaluated by fluorescence microscopy. Unstimulated confluent HUVECs were shown to bind minimally to U937 cells (see FIGS. 6A and 6B). Endothelial adhesion to U937 increased substantially 2.95-fold when HUVECs were treated with TNF-α at 20 ng / ml for 12 hours (see Figures 6A and 6B). Preculture (25 μg / ml, 6 hours) of HUVECs with α-iso-cubebene showed a significant reduction in U937 adhesion to HUVECs, for example TNF-α (20 ng) as shown in FIGS. 6A and 6B. / ml, 12 hours) was reduced from 2.95 times to 1.34 times.

Example 3-7: Electrophoretic Mobility Shift Assay (EMSA)

HUVECs were pretreated with α-iso-cubebene (25 μg / ml) for 6 hours and then stimulated with TNF-α (20 ng / ml) for 30 minutes and 60 minutes at 37 ° C. prior to extraction of nuclear proteins. The NF-κB oligonucleotide (5'-AGTTGAGGGGACTTTCCCAGGC-3 ') (Bioneer) was terminally labeled with biotin. The nuclear protein extract (10 μg) was added to a 4% non-modified polyacrylamide gel and the gel was operated at 100 V in a 0.3X TBE buffer at room temperature until the bromophenol blue dye reached the bottom of the gel. Prior to incubation for 20 minutes with 20 fmol of biotin-labeled NF-κB oligomernucleotides. When the electrophoretic finish, the gel were detected as Biodyne ^ⓡ B Nylon Membrane (Pierce) and the blotting, the screen label oligonucleotide photoelectric the chemiluminescence according to the manufacturer's instructions for the nucleotide (Pierce) kit to EMSA. Images were obtained and quantified using the LAS-3000 imaging system (Fujifilm, Tokyo, Japan).

Attenuation of TNF-α-induced NF-κB activity in HUVEC by α-iso-cubebene

 TNF-α-induced nuclear expression of NF-κB in HUVECs was not detected under control conditions. EMSA results showed NF-κB shifted bands when treated with TNF-α (20 ng / ml, 37 ° C. for 30 and 60 minutes) (see FIG. 9). α-iso-cubebene (25 μg / ml) significantly reduced the intensity of TNF-α-induced NF-κB migrated bands (39.8% inhibition at 30 minutes and 26.2% inhibition at 60 minutes; P <0.01, respectively) And P <0.05).

 From these results, it was confirmed that α-iso-cubebene inhibited the activity of NF-κB transcription factor induced by TNF-α.

Example 3-8 Determination of Reactive Oxygen Species (ROS) Production

ROS has been shown to activate transcriptional endothelial transcription factors in culture and has been suggested as a common second messenger for various pathways leading to NF-κB activity. The effect of α-iso-cubebene on ROS production in HUVECs was determined by fluorescence microscopy using 2,7-dichlorofluorescein diacetate (DCFH-DA, Sigma) as a probe for the presence of H ₂ O ₂ . . HUVECs (2 × 10 ⁴ cells / well) in 24-well plates were pretreated with α-iso-cubebene (25 μg / ml) for 6 hours and TNF-α (20 ng / ml) was added to the medium for 60 minutes. After removing the medium from the wells, the cells were incubated with 20 μM DCFH-DA for 30 minutes. Labeled cells were examined under an Olympus BX50 microscope equipped with fluorescence epi-illumination. Micrographs were obtained electronically at 1,360 x 1,024 pixel resolution using an Olympus DP70 digital camera. The results obtained are expressed as mean ± standard deviation. The results were analyzed statistically by two-tailed t-test. Significant difference was P <0.05.

Inhibition of TNF-α-induced ROS generation by α-iso-cubebene

 As shown in FIG. 7, stimulation with 20 ng / ml TNF-α significantly increased intracellular ROS production in HUVECs (see FIG. 7). Treatment with α-Iso-cubebene (25 μg / ml) for 6 hours was able to significantly inhibit TNF-α-induced ROS formation in HUVECs.

Α-iso-cubebene compound isolated from the Schizandra chinensis of the present invention can be confirmed that not only purely separated highly active natural substances but also has an excellent effect on anti-inflammatory.

Claims (11)

1. [Revision 25.06.2009 under Rule 26]
   Α-iso-cubebene compound having the following Chemical Formula 1 extracted from Schisandra chinensis , Chemical Fomula C ₁₅ H ₂₄ , and having a molecular weight of 204.35. [Formula 1]

   
2.  A composition for the prevention and treatment of anti-inflammatory diseases containing the compound of claim 1 as an active ingredient.
3.  The method of claim 2, wherein the anti-inflammatory disease is atherosclerosis, diabetes, arthritis, obesity, inflammatory bowel disease (IBD), Alzheimer's disease, multiple sclerosis, tuberculosis, sarcoidosis ), Hepatitis, cholecystitis, fungal infections, gastric ulcers, asthma, atopic dermatitis, tendinitis, or nephritis.
4.  A method of expressing anti-inflammatory properties by administering to a patient in need of the prophylactic and therapeutic composition according to claim 2 or 3 to inhibit the expression of the cell surface adhesion molecule VCAM-1 increased by TNF-α in vascular endothelial cells.
5.  A method of expressing anti-inflammatory properties by administering to a patient in need of the prophylactic and therapeutic composition of claim 2 or 3 to inhibit the expression of the cell surface adhesion molecule E-selectin increased by TNF-α in vascular endothelial cells.
6.  A method of expressing anti-inflammatory properties by administering to a patient in need of the prophylactic and therapeutic composition of claim 2 or 3 to inhibit the expression of pro-inflammatory cytokine MCP-1 increased by TNF-α in vascular endothelial cells.
7.  A method for expressing anti-inflammatory properties by administering to a patient in need of the prophylactic and therapeutic composition of claim 2 or 3 to inhibit the expression of proinflammatory cytokine IL-6 increased by TNF-α in vascular endothelial cells.
8.  A method of expressing anti-inflammatory properties by administering to a patient in need of the prophylactic and therapeutic composition of claim 2 or 3 to inhibit the expression of pro-inflammatory cytokine IL-8 increased by TNF-α in vascular endothelial cells.
9.  A method of expressing anti-inflammatory properties by inhibiting adhesion between vascular endothelial cells and leukocytes by administering to a patient in need of the prophylactic and therapeutic composition of claim 2 or 3.
10.  A method of expressing anti-inflammatory properties by administering to a patient in need of the prophylactic and therapeutic composition of claim 2 or 3 to control inflammation through the activity of vascular endothelial cells.
11.  Obtaining a hexane extract from the schisandra chinensis using hexane;

     Adding the hexane extract to the surface of the column filled with silica gel, and sequentially adding hexane, hexane-ethylacetic acid and chloroform-methanol mixed solution to the column to obtain a plurality of fractions;

    Adding a first fraction of the fractions to a column packed with silica gel to obtain a subsequent fraction while flowing a CH ₂ Cl ₂ solution; And

    Adding the resultant fraction to a silica gel-filled column to separate the pure water with flowing hexane solution to identify α-iso-cubebene compound having Chemical Fomula C ₁₅ H ₂₄ , molecular weight 204.35; Method for extracting α-iso-cubebene compound from Schizandra consisting of

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