KR20090070188A - Extracts of cirsium japonicum and uses thereof - Google Patents

Abstract

A Cirsium japonicum extract having antioxidative activity and anti-inflammatory activity is provided to suppress the activity of hyalurodinase and collagenase and prevent and treat rheumatoid arthritis and atherosclerosis. A Cirsium japonicum extract is extracted with ethanol, methanol, propanol, chloroform or acetone. The Cirsium japonicum extract is obtained by drying, pulverizing, adding ethanol per 30g of pulverized sample, and maintaining at room temperature. The active amount of the Cirsium japonicum extract is 0.1-10 mg/kg, and the extract is administered one to three times a day. A pharmaceutical composition for preventing or treating rheumatoid arthritis and atherosclerosis comprises the Cirsium japonicum extract.

Description

Thistle extract and its uses {Extracts of Cirsium japonicum and uses apparent

The present invention relates to thistle extract with antioxidant and anti-inflammatory activity and use thereof. Specifically, the present invention relates to a pharmaceutical composition for the prevention or treatment of rheumatoid arthritis or atherosclerosis, including thistle extract having antioxidant activity and anti-inflammatory activity.

Rheumatoid arthritis is an inflammatory disease in which the body's immune system attacks the synovial membrane that covers the joints, and over time, the synovial membrane becomes large, damaging ligaments, cartilage and bones.

Free radical species are a problem associated with the development of rheumatoid arthritis. Reactive oxygen species primarily stimulate and overexpress oxidative stress as well as induced nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) associated with rheumatoid arthritis. In addition, reactive oxygen species cause intercellular adsorption of monocytes and human umbilical vein endothelial cells (HUVECs) in immune inflammatory reactions, and cellular adhesion molecules (CAMs). Causes the expression of. Intercellular adsorption of mononuclear leukocytes and HUVECs occurs in the early stages of immune inflammation. CAM is stimulated by the inflammation mediator TNF-α to increase expression and increase the intercellular adsorption of mononuclear leukocytes and HUVECs, leading to rheumatoid arthritis as well as atherosclerosis.

In addition, hyaluronidase, which is effective in treating arthritis and promotes the hydrolysis of hyaluronic acid, which is part of cartilage synovial fluid, and collagenase, which is directly involved in the degradation of collagen and proteoglycan cartilage tissue of joints. Increased activity can also increase the incidence of rheumatoid arthritis.

Meanwhile, thistle ( Cirsium japonicum ) is an asteraceae plant, a herbaceous plant native to Korea, Japan, and China. Medically thistle has been used to treat high blood pressure, diuresis and hepatitis. In addition, thistle has been used for the treatment of cancers such as liver cancer, kidney cancer, blood cancer, etc., and the anti-anxiety function and cell division inhibitory activity of thistle has been reported. In addition, the antibacterial activity of thistle flower and leaf extracts was superior to other extracts, and the growth inhibition and sterilization activity of the bacteria were reported for gram positive bacteria.

Recently, the pharmacological effects of traditionally used medicinal plants have been revealed and used in medicine or functional foods are increasing. The inventors of this invention inhibit the activity of hyaluronidase and collagenase, inhibit the production of LPS-induced nitrite, VCAM-1 (vascular cellular adhesion molecule-1) in CAM, By inhibiting intracellular adhesion molecule-1 (ICAM-1) and E-selectin transcription, it inhibits the intercellular adsorption of HUVECs and mononuclear leukocytes, thereby confirming its effectiveness in preventing or treating rheumatoid arthritis or atherosclerosis. The present invention has been completed.

It is an object of the present invention to provide a new plant extract comprising a substance having antioxidant and anti-inflammatory activity and a pharmaceutical composition comprising the same.

In order to achieve the above object, the present invention provides thistle extract with antioxidant or anti-inflammatory activity.

The present invention also provides a pharmaceutical composition comprising thistle extract.

Hereinafter, the present invention will be described in detail.

The present invention provides an antioxidant extract and an anti-inflammatory extract isolated from thistle using solvent extraction.

As a solvent used in the solvent extraction method, an organic solvent such as ethanol, methanol, propanol, chloroform, acetone can be used, and preferably ethanol is used.

When ethanol is used as a solvent, thistle is dried and ground, and 100 ml of ethanol is added per 30 g of the ground powder sample, followed by extraction while standing at room temperature.

When separating the said extract, it is preferable to use a leaf part among thistles.

The present invention also provides a pharmaceutical composition comprising thistle extract.

Thistle extract contained in the pharmaceutical composition is preferably used thistle leaf extract separated by the solvent extraction method.

The pharmaceutical composition may be administered orally or parenterally during clinical administration and may be used in the form of a general pharmaceutical preparation. In addition, the pharmaceutical composition may be administered in various oral and parenteral dosage forms in actual clinical administration, and when formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc., which are commonly used It is prepared using. Solid form preparations for oral administration include tablets, pills, powders, granules, capsules and the like, and such solid form preparations include at least one excipient such as starch, calcium carbonate, sucrose or lactose in the floral fragrance extract of the present invention. Mixed with gelatin. In addition to the excipients, lubricants such as magnesium styrate and talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water, liquid, and paraffin. have. Formulations for parenteral administration include sterile aqueous solutions, secretarial solvents, suspensions, emulsions, lyophilizers, suppositories, and the like. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate and the like can be used. As the base of the suppository, utopsol, macrogol, tween 61, cacao butter, laurin butter, glycerol gelatin and the like can be used.

The effective amount of thistle extract in the pharmaceutical composition of the present invention is 0.1-10 mg / kg, the formulation of the pharmaceutical composition comprising thistle extract may be administered 1-3 times a day.

Hyaluronidase (HAase) is an enzyme involved in the immune response and is an enzyme that hydrolyzes hyaluronic acid. The hyaluronic acid is a part of the synovial fluid secreted from the joints to soften the cartilage and joints that buffer the bones between the bones, and in the cartilaginous tissues to help bond between molecules to reinforce the cartilage to withstand joint movement and weight well. Play a role. In addition, collagenase, a matrix metalloproteinase-1 (MMP-1), cleaves collagen types I and III, which are directly involved in the degradation of collagen and proteoglycan cartilage tissue in joints. Extract of the present invention can treat or prevent rheumatoid arthritis by inhibiting the activity of such hyaluronidase and collagen degrading enzyme.

Nitric oxide is associated with inflammatory joint disease, acts as an important factor in catabolism mediated by inflammation, and is an important regulator in the progression of the inflammatory response in rheumatoid arthritis. The resulting nitric oxide also induces the expression of cyclooxygenase-2 (COX-2) in blood cells. The extract of the present invention can treat or prevent rheumatoid arthritis by inhibiting the production of nitrite induced by lipopolysaccharide (LPS).

Exposure of human umbilical vein endothelial cells (HUVECs) in blood vessels to inflammatory mediators such as TNF-α increases the expression of cellular adhesion molecules (CAMs) for adsorption of HUVECs and monocytes. This leads to rheumatoid arthritis and atherosclerosis. The extract of the present invention can treat or prevent rheumatoid arthritis or atherosclerosis by inhibiting the transcription of VUV-1, ICAM-1, E-selectin in CAM and inhibiting the intercellular adsorption of HUVECs and mononuclear leukocytes.

Thistle extract of the present invention inhibits the activity of hyaluronidase and collagenase, inhibits the production of LPS-induced nitrite, inhibits CAM transcription and inhibits intercellular adsorption of HUVECs and mononuclear leukocytes, It can be used as an active ingredient in a pharmaceutical composition for preventing or treating arthritis, in particular rheumatoid arthritis or atherosclerosis. In addition, thistle extract of the present invention can be easily produced without chemical synthesis, economical, high safety because there is little toxicity to cells.

The invention is illustrated by the following examples. However, the present invention is not limited by the following examples.

Example  1: Preparation of thistle leaf extract

Thistle leaves were washed, dried with a lyophilizer, and then crushed. To 30 g of crushed leaf powder was added 100 ml of 70% ethanol and left at room temperature for 24 hours. The resulting mixture was filtered through Whatman No. 1 filter paper to remove impurities. Thistle extract was prepared by concentrating the obtained extract using a distillation machine under reduced pressure.

Experimental Example  1: of extract DPPH Radical  Scavenging activity

0.2 ml of methanol solution containing thistle extract (500-2,000 μg / mL), 4 ml of methanol and DPPH (2,2-diphenyl-1-picrylhydrazyl) (Sigma (Sigma-Aldrich Inc., St. Louis) , MO, USA)) 0.5 mL of methanol solution (1 mmol / L) was added. The resulting mixture was stirred for 15 seconds and kept at room temperature for 30 minutes. The DPPH radical scavenging activity was investigated by measuring absorbance at 517 nm using a UV-spectrometer (Agilent Technologies Inc., Santa Clara, Calif., USA). The case where thistle extract was not treated was used as a control, and the absorbance was measured in the same manner as above. In order to compare the DPPH radical scavenging activity of thistle extract with a known antioxidant ascorbic acid (Asc, 1,000 μM), the absorbance was measured in the same manner as above for ascorbic acid. DPPH radical scavenging activity was calculated as the ratio of control group not treated with thistle extract. The results are shown in FIG.

As shown in Figure 1, DPPH radical scavenging activity increased with the treatment concentration of thistle leaf extract. When thistle leaf extracts were treated at 1000 μg / ml and 2000 μg / ml concentrations, they exhibited about 64% and 82% radical scavenging activity, respectively. When thistle leaf extract was treated at a concentration of 1000 ㎍ / ml, DPPH radical scavenging activity was in vivo ( in vivo ) and in vitro ( in It was similar to ascorbic acid used as antioxidant.

Experimental Example  2: reducing power of extract

Thistle extract was dissolved in distilled water to form an aqueous solution of 10-1000 ug / ml, mixed with 2.5 mL of 0.2 M phosphate buffer (pH 6.6) and 2.5 mL of 1% K ₃ Fe (CN) ₆ , and then heated to 50 ° C. for 20 minutes. Reaction at Then 2.5 mL of 10% trichloroacetic acid was added and centrifuged for 10 min at 2,090 × g. 2.5 mL of the obtained supernatant was mixed with 2.5 mL of distilled water and 0.5 mL of 0.1% FeCl ₃ . Absorbance was measured at 700 nm using a UV-spectrometer (Agilent Technologies Inc., Santa Clara, Calif., USA). The results are shown in Table 1 below.

Table 1: Reducing Power of Extracts

Extract Treatment Concentration (㎍ / ml) Reducing power 12.5 ND ^{1 )} 25 0.01 ± 0.00 50 0.13 ± 0.00 100 0.05 ± 0.00 500 0.19 ± 0.01 1000 0.40 ± 0.00

1) ND: No measurement

The reducing power of thistle leaf extract increased with increasing concentration of extract. This result was consistent with the DPPH radical scavenging activity result.

Experimental Example  3: Hyaluronidase ( HAase Inhibitory activity

HAase inhibitory activity Elson-Morgan method (Lee J, Lee SH, Min KR, Lee KS, Ro JS, Ryu JC, Kim Y. Inhibitory effects of hydrolysable tannins on Ca2 + -activated HAase Planta Med 59:... 381-382 (1993)).

HAase solution (EC 3.2.1.35) (Sigma-Aldrich Inc., St. Louis, MO, USA) (7,900 units / mL in 0.1 M acetate buffer, pH 3.5) (50 μl) and thistle extract DMSO solution (500 Or 1000 μg / mL) 100 μl were mixed and incubated at 37 ° C. for 20 minutes. Then, 0.1 ml of 12.5 mM CaCl ₂ , an HAase activator, was added and incubated again at 37 ° C. for 20 minutes. Then 250 μl of hyaluronic acid (Fluka (Sigma-Aldrich Inc., St. Louis, Mo., USA)) (1.2 mg / mL solution in 0.1 M acetate buffer, pH 3.5) was added and 20 minutes at 37 ° C. Incubated for 0.1 mL of 0.4 N NaOH and 0.1 mL of 0.4 M potassium tetraborate were added and the HAase reaction was stopped by heating at 100 ° C. for 3 minutes. After the reaction solution was completely cooled, 3 mL of DMAB solution (4 g of dimethylaminobenzaldehyde, 350 mL of 100% glacial acetic acid, and 50 mL of 10 N HCl) was added and reacted at 37 ° C. for 20 minutes. The reaction product, 4-acetylglucosamine, was measured for absorbance at 585 nm using a UV-spectrometer (Agilent Technologies Inc., Santa Clara, Calif., USA). As a control, a 1% DMSO solution containing no thistle extract was used.

HAase inhibitory activity was expressed as a percentage decrease in absorbance compared to the control, was calculated using the following equation, the results are shown in Table 2 below.

HAase inhibitory activity = [(Ac-As) / Ac] × 100

Ac: absorbance of the control group

As: absorbance of the sample

Table 2: HAase Inhibitory Activity

Extract treatment concentration Inhibitory Activity (%) 500 μg / ml 62.00 ± 4.30 1000 μg / ml 60.70 ± 9.87

When the thistle leaf extract concentration was increased, the amount of reaction product produced decreased, indicating a HAase inhibitory activity.

Experimental Example  4: Collagen Inhibitory Activity

Collagen inhibitory activity was modified by Sawabe's method (Sawabe Y, Yamasaki K, Iwagami S, Kazimura K, Nakagommi K. Inhibitory effects of natural medicines on the enzymes related to the skin.Yakugaku Zasshi 118: 423-429 (1998) Was used.

Collagen degrading enzyme (EC 3.4.24.3) (Sigma (Sigma-Aldrich Inc., St. Louis, MO, USA)) 5.0 μg, PZ-peptide (Fluka (Sigma-Aldrich Inc., St. Louis, MO, USA) ) 0.5 mg, and thistle leaf extract (500 μg / ml and 1000 μg / ml) were mixed in 0.1 M Tris buffer (pH 7.4, total volume 1.7 mL). The resulting mixture was incubated at 37 ° C. for 30 minutes, after which 1 ml of 25 mM citric acid solution was added to stop the enzyme reaction. 5 ml of ethyl acetate were added, and the absorbance was measured at 320 nm using a UV-spectrometer (Agilent Technologies Inc., CA, USA) using an organic solvent layer. In the case where only thistle leaf extract is included without the addition of collagenase, the same method is performed for the case where the collagenase is added and thistle leaf extract is not included, and when both the collagenase and thistle extract are not included. The absorbance was measured.

The percentage of collagenase activity inhibition was calculated by the following equation, and the results are shown in Table 3.

Collagen degrading enzyme activity (%) = [(Ac-As) / Ac] × 100

Ac: Absorbance without the addition of collagenase and thistle leaf extract-Absorbance without both the collagenase and thistle extract

As: Absorbance with the addition of thistle leaf extract and collagenase-Absorbance with the addition of collagenase and without thistle leaf extract

Table 3: Inhibition of Collagenase Activity

Extract treatment concentration Inhibitory Activity (%) 500 μg / ml 0.79 ± 19.9 1000 μg / ml 31.86 ± 7.78

Thistle leaf extract showed collagenase inhibitory activity.

Experimental Example  5: cytotoxicity investigation

(1) cell culture

RAW 264.7 murine macrophage cells (American Type Culture Collection (ATCC), Manassas, VA, USA) were treated with 10% FBS, penicillin (100 units / mL) / streptomycin (100 μg / mL) (GIBCO (Invitrogen Inc. , Grand Island, NY, USA)) was incubated using a humidified incubator at 37 ℃, 5% CO ₂ in DMEM medium containing.

HUVEC (CRL-2480, ATCC, Manassas, VA, USA) was added to 10% FBS (GIBCO (Invitrogen Inc., Grand Island, NY, USA)), penicillin / streptomycin (GIBCO (Invitrogen Inc., Grand Island, NY, USA). USA)) F-12K nutrient mixtures containing 100 units / mL, 0.1 mg / mL heparin and 0.03 mg / mL ECGS (endothelial cell growth supplement) ((GIBCO (Invitrogen Inc., Grand Island, NY, USA)) F-12K nutrient mixture) (Kaighn 's modification, GIBCO, Invitrogen Inc.) in cultured at 37 ℃, 5% CO ₂ humidified incubator. HUVEC plasma containing trypsin inhibitor was washed twice with a single layer in PBS (pH 7.4) The components were removed and passaged using 0.05% Trypsin-EDTA (0.53 mM EDTA) ((GIBCO (Invitrogen Inc., Grand Island, NY, USA)).

(2) cytotoxicity investigation

96-well tissue culture plates (Corning Inc., Corning, NY, USA) were seeded with the cultured RAW 264.7 cells and HUVEC at a concentration of 1 × 10 ⁵ cells / well, respectively, and RAW 264.7 cells were 3-4 hours old, HUVEC was incubated at 37 ° C. for 24 hours and adsorbed to the bottom. Thistle leaf extract was added 50-500 μg / ml and incubated for 24 hours. 0.5 mg / MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide) (Sigma (Sigma-Aldrich Inc., St. Louis, Mo., USA)) Each well was added at a concentration of mL and continued incubation at 37 ° C. for 4 hours.

The medium of each plate was removed and 100 μl of dimethyl sulfoxide (DMSO) was added to each well and incubated at room temperature for 5 minutes. The resulting formazan was measured for absorbance at 540 nm using a UV-spectrophotometric plate reader (Emax, Molecular Devices Inc., Sunnyvale, Calif., USA). The control group was measured for absorbance in the same manner except that thistle leaf extract was not treated. Cell viability was expressed as a percentage of absorbance of the control and group treated with thistle leaf extract. Survival of RAW 264.7 cells is shown in FIG. 2. The survival rate of HUVEC is shown in FIG. 3.

As shown in FIG. 2, no toxicity was observed for RAW 264.7 cells in thistle leaf extract 0-1000 μg / ml. Thus, thistle leaf extract 25-1000 μg / ml was used for nitrite production inhibition assay.

As shown in Figure 3, thistle leaf extract showed less toxicity (> 80% cell viability) against HUVEC monolayers at all treatment concentrations ((0-5,000 μg / mL). It was found to be non-toxic to the cell proliferation of HUVEC monolayers involved in intercellular adsorption, the first stage of the inflammatory response, so thistle leaf extracts 500, 1000, and 5000 μg / mL were tested for cell adsorption using HUVEC monolayers. Used.

Experimental Example  6: nitrite analysis

(1) RAW 264.7 cell culture

RAW 264.7 rat macrophage cells (American Type Culture Collection (ATCC), Manassas, VA, USA) were used in 10% FBS (GIBCO (Invitrogen Inc., Grand Island, NY, USA)), penicillin (100 units / mL) / streptomycin Incubated in a DMEM medium containing (100 μg / mL) (GIBCO (Invitrogen Inc., Grand Island, NY, USA)) using a humidified incubator at 37 ° C., 5% CO ₂ .

(2) nitrite analysis

The cultured RAW 264.7 cells were incubated at 37 ° C. for 3-4 hours using a 5% CO ₂ humidified incubator at a concentration of 2 × 10 ⁵ cells / well in 96 well culture plates. Nitrite production was stimulated by adding 1 μg / ml of LPS (Sigma (Sigma-Aldrich Inc., St. Louis, Mo., USA)) to RAW 264.7 cells attached to the bottom of the well plate. Then, thistle leaf extract 25-1000 μg / ml was added and incubated for 24 hours. The nitrite generated induced in RAW 264.7 cells by LPS Griess reaction (Chattopadhyay S, Bhaumik S, Purkayastha M, Basu S, Chaudhuri AN, Gupta SD. Apoptosis and necrosis in developing brain cells due to arsenic toxicity and protection with antioxidants. Toxicol .. Lett 136: 65-76 was measured in the (2002)). Specifically, the mixture was reacted by mixing 100 µl supernatant with 100 µl of Griess reagent (1% sulfanamide in 5% phosphoric acid and 0.1% N-1-naphthylethylenediamine dihydrochloride), followed by a microplate reader ( Absorbance was measured at 540 nm using Emax, Molecular Devices Inc.). As a control, absorbance was measured in the same manner except that LPS was not treated and thistle extract was not treated. The nitrite production inhibition rate is expressed as the nitrite production rate relative to the control. The results are shown in FIG.

As shown in Figure 4 thistle extract inhibited the production of nitrite depending on the treatment concentration. Thistle leaf extract partially inhibited nitrite production in proportion to the concentration of 250-1,000 μg / mL extract. 1,000 μg / ml thistle leaf extract reduced nitrite production by about 70%.

Experimental Example  7: THP -1 and HUVEC Intercellular  Adsorption inhibition

(1) calcein-AM labeling of mononuclear leukocytes

The mononuclear leukocyte cells THP-1 (Korean Cell Line Bank (KCLB), Seoul, South Korea) were prepared in 37 ° C., 5% CO ₂ incubator in RPMI-1640 medium containing 100% / mL of 10% FBS and penicillin / streptomycin. Incubated. Then, the cells were recovered by centrifugation at 2,090 × g for 2 minutes and then subcultured. 5 μM calcein-AM (calcin O, O -diacetate tetrakis (acetoxymethyl) ester) Sigma (Sigma-Aldrich Inc., St. Louis, MO, USA) (PBS solution, pH) in cultured mononuclear leukocyte cells 7.4) was added and incubated at 37 ° C. for 30 minutes. Then, washed three times with PBS to label and remove the remaining calcein-AM.

(2) cell adsorption assay

HUVECs cultured in Experimental Example 5 in 96 well tissue culture plates (Corning 3603, Corning Inc.) were seeded at a concentration of 1 × 10 ⁵ cells / well and incubated at 37 ° C. for 24 hours. Milk thistle extract 500, 1000, 5000 ㎍ / ml was added to the HUVEC monolayer and incubated for 24 hours. Then 5 ng / mL of TNF-α (tumor necrosis factor-α) (BD Science (San Jose, Calif., USA)) was added and cell adsorption was activated for 24 hours. Washed three times with PBS, THP-1 labeled with Calcein-AM was mixed with activated HUVEC at a concentration of 5 × 10 ⁵ cells / well and incubated in a 37 ° C., 5% CO ₂ humidified incubator for 1 hour. . It was then removed by 4 washes with PBS to remove unadsorbed THP-1. Adsorption of THP-1 labeled with HUVEC and Calcein-AM was measured using a fluorescence plate reader (FL600, Bio-Tek Instruments, Inc., Winooski, VT, USA). 485 nm and 530 nm were used as excitation / emitting wavelengths of Calcein-AM molecules. For comparison of the adsorption inhibition effect, the control group was treated only with TNF-α and the absorbance was measured without thistle leaf extract. The results are shown in FIG.

As shown in FIG. 5A, the inhibition of adsorption between THP-1 and HUVEC cells was not observed in 500 ㎍ / ml thistle leaf extract. However, thistle leaf extract inhibited about 82% of intracellular adsorption at 1,000 μg / mL and at about basal level at 5,000 μg / mL.

For cell adsorption photos, HUVECs were cultured in the same manner as above in 24 well culture plates, and the cultured HUVECs were adsorbed with THP-1 labeled with Calcein-AM in the same manner as above. Adsorption of Calcein-AM labeled THP-1 and HUVEC was recorded using an inverted fluorescence microscope (IX 71, OLYMPUS Inc., Tokyo, Japan) at 100 and 200 magnifications. Observed in real time using an OLYMPUS DP50 camera attached to a microscope (Imaging software, ViewfinderLite, Ver. 1.0.134, Pixera Corporation, Los Gatos, CA, USA, OLYSIA BioAutoCell Ver. 3.2, Soft Imaging System, Tokyo, Japan) , Confirmed with a photo. The results are shown in FIG.

5B shows representative images of enhancement and inhibition of TNF-α-induced intercellular adsorption by the extract of thistle leaf. Thistle leaf extracts inhibited TNF-α-induced intercellular adsorption labeled with calsein-AM at baseline levels.

Experimental Example  8: CAM  Protein transcription RT - PCR  analysis

RNA was extracted from HUVEC treated with thistle extract using the RNeasy kit (Qiagen Inc., Valencia, CA, USA). RT-PCR was performed using a One-Step RT-PCR kit from Qiagen and Bioneer. The primers used for PCR, VCAM-1, ICAM-1, and E-selectin primers, and the control gene GAPDH (glyceraldehyde-3-phosphate dehydrogenase) primers are shown in Table 4 below. Primers were used at a final concentration of 1 μM.

Table 4. RT-PCR primers (FP: forward primer, RP: reverse primer)

gene primer VCAM-1 (FP) 5'-ATGCCTGGGAAGATGGTCGTGA-3 ' VCAM-1 (RP) 5′-TGGAGCTGGTAGACCCTCGCTG-3 ′ ICAM-1 (FP) 5′-GGTGACGCTGAATGGGGTTCC-3 ICAM-1 (RP) 5′-GTCCTCATGGTGGGGCTATGACTC-3 ′ E-selectin (FP) 5′-ATCATCCTGCAACTTCACC-3 ′ E-selectin (RP) 5′-ACACCTCACCAAACCCTTC-3 ′ GAPDH (FP) 5′-ATGACAACAGCCTCAAGATCATCAG-3 ′ GAPDH (RP) 5′-CTGGTGGTCCAGGGGTCTTACTCCT-3 ′

As a control, thistle extract and RNA of HUVEC before TNF-α treatment (1 μg / μL) were used, and GAPDH transcription was examined to investigate the efficiency of PCR. Bio-Rad thermal cycler (MJ Mini, Bio-Rad Inc., Hercules, Calif., USA) was used, and RT reaction was performed at 50 ° C. for 30 minutes for cDNA synthesis and predenaturation. , 15 minutes at 95 ° C. PCR amplification was performed 30 times with denaturation at 95 ° C. for 1 minute, annealing at 55 ° C. for 2 minutes, and extension at 72 ° C. for 3 minutes. Finally, a final extension was made at 72 ° C. for 10 minutes. RT-PCR products were separated on agarose gels. The results are shown in Figs. 6 to 8 (Figs. 6B, 7B, and 8B).

In addition, separation of the agarose gel was performed three times independently of each other, and the concentration of each band was quantified using densitometric software (Quantity one, version 4.6.3., Bio-Rad.,). The results are shown in FIGS. 6 to 8 (FIGS. 6A, 7A, and 8A).

As shown in Figures 6-8, the addition of TNF-α significantly increased the transcription of CAM proteins of VCAM-1, ICAM-1, E-selectin. However, when thistle leaf extract was treated, the decrease in transcription of mRNA of CAM protein increased with the concentration of thistle extract. Electrophoresis of RT-PCR results on the CAM protein of HUVEC cells showed no DNA bands. In particular, thistle leaf extract as shown in Figure 6 effectively inhibited the increase of transcription by TNF-α of VCAM-1 by about 500 μg / mL addition. On the other hand, as shown in Figures 7 to 8, in the case of ICAM-1 and E-selectin mRNA transcription, RT-PCR band faded as the concentration of the extract increased, using an extract of 5,000 ㎍ / mL Transcription decreased to basic levels when treated.

1 shows DPPH radical scavenging activity and DPPH radical scavenging activity of ascorbic acid (Asc) according to the concentration of thistle leaf extract.

Figure 2 shows the survival rate of RAW 264.7 cells according to the concentration of thistle leaf extract.

Figure 3 shows the survival rate of HUVEC according to the concentration of thistle leaf extract.

Figure 4 shows the production rate of nitrite induced by LPS according to the concentration of thistle leaf extract.

Figure 5 shows the intercellular adsorption inhibition effect of THP-1 and HUVEC according to the concentration of thistle leaf extract, A is TNF-α only inhibit the intercellular adsorption of THP-1 and HUVEC according to the treatment concentration of thistle leaf extract Compared to the treated case, B represents the fluorescence image according to each experimental case.

Figure 6 shows the transcriptional inhibitory effect of VCAM-1 according to the concentration of thistle leaf extract, A is the case of treatment of RNA transcription of VCAM-1 according to the treatment concentration of thistle leaf extract with only the basic level and TNF-α B is the RNA band in the agarose gel for each experimental case.

Figure 7 shows the effect of inhibiting the transcription of ICAM-1 according to the concentration of thistle leaf extract, A is the case of treatment of RNA transcription of ICAM-1 according to the treatment concentration of thistle leaf extract with only the basic level and TNF-α B is the RNA band in the agarose gel for each experimental case.

Figure 8 shows the transcriptional inhibitory effect of E-selectin-1 according to the concentration of thistle leaf extract, A is the basic level and TNF-α only inhibits RNA transcription of E-selectin-1 according to the treatment concentration of thistle leaf extract Compared to the treated case, B represents the RNA band in the agarose gel according to each experimental case.

Claims (6)

Thistle Cirsium Thistle extract with antioxidant activity isolated from japonicum ) . Thistle extract with anti-inflammatory activity isolated from thistle. The thistle extract according to claim 1 or 2, wherein the thistle extract is isolated from thistle leaf. The thistle extract according to claim 1 or 2, wherein the thistle extract is selected from the group consisting of ethanol, methanol, propanol, chloroform and acetone. Pharmaceutical composition comprising the thistle extract of claim 1 or 2 as an active ingredient. The pharmaceutical composition of claim 5, wherein the pharmaceutical composition is for preventing or treating rheumatoid arthritis or atherosclerosis.

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