KR20090068560A - Extracts from allium victorialis subsp. platyphyllum having anti-inflammatory activity - Google Patents

Abstract

A composition containing extracts of Allium victorialis subsp. which has anti-inflammation activity is provided to prevent and treat rheumatoid arthritis or atherosclerosis. An extract from Allium victorialis subsp. having antioxidation and anti-inflammation activities is obtained by extracting an Allium victorialis subsp. with ethanol, methanaol, propanol, chloroform, and acetone. The extract of Allium victorialis subsp. Is obtained from leaves or seed of Allium victorialis subsp. A pharmaceutical composition for preventing or treating anti-inflammatory disease contains the extract of Allium victorialis subsp. as an active ingredient. The composition is used in a form of solid formulation such as tablet, powder, pill, granule, and capsule, or parenteral administration such as sterilized solution, suspension, emulsion, and suppository.

Description

Mountain garlic extract having anti-inflammatory activity {Extracts from Allium victorialis subsp. platyphyllum having anti-inflammatory activity

The present invention relates to an acid garlic extract having anti-inflammatory activity and a composition comprising the same.

Rheumatoid arthritis is chronic inflammation of the synovial membrane of the joints in the human body. Once rheumatoid arthritis begins, it forms a `` Pannus '' clump of inflammatory cells from the blood of the synovial tissue, which destroys the cartilage, causes joint deformation and weakens the bones around the joint. do. This disease is an abnormal function of the body's immune function, occurs because the immune system that acts to defend the body against foreign substances such as bacteria in the body attack their joints for unknown reasons. You can get rheumatoid arthritis regardless of age or gender, but it usually occurs in people in their 30s and 40s.

In order to treat such rheumatoid arthritis, aspirin and nonsteroidal anti-inflammatory drugs, low dose oral steroids, antirheumatic drugs, and intra-articular steroid injections are used. However, if it is possible to prevent or treat rheumatoid arthritis through the ingestion of food in daily life in addition to these drug-dependent therapies, this would be of great help to the patient.

Mountain Garlic ( Allium victorialis subsp. platyphyllum ) is a perennial herb belonging to the monocotyledonous genus Liliumaceae, and is distributed throughout the region. The extract of each part of acid garlic is edible, and has been used traditionally for herbal medicines such as cold, antibacterial, digestive and dry stomach. In addition, sulfur compounds and antiplatelet aggregation activity with potential antithrombotic activity have recently been reported. In addition, the effect of the acid garlic extract has been widely studied, liver composition containing the acid garlic extract as an active ingredient or a composition for the prevention and treatment of liver disease (Korean Patent No. 2005-102572), acid garlic extract as an active ingredient A composition for the prevention and treatment of diabetic diseases (Korean Patent Laid-Open No. 2005-43092), and a topical skin composition (Korean Patent Laid-Open No. 2006-12805) containing an active garlic extract have been reported.

The inventors of the present invention separated the extract by solvent extraction using acid garlic, and completed the present invention by revealing that the obtained extract has antioxidant activity and anti-inflammatory activity.

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

In order to achieve the above object, the present invention provides an antioxidant and anti-inflammatory extract of acid garlic.

In addition, the present invention provides a pharmaceutical composition and functional food comprising a mountain garlic extract.

Hereinafter, the present invention will be described in detail.

The present invention provides an extract obtained from acid garlic using a solvent extraction method. The solvent extraction method is a method of obtaining an extract in a conventional manner by adding a solvent to a portion of the acid garlic. 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 the solvent, the solvent extraction method is preferably dried by grinding a predetermined portion of the acid garlic, and extracted using 100 ml of ethanol per 30 g of the acid garlic powder obtained by grinding.

In order to obtain an extract from the acid garlic, roots, seeds, leaves, flower stalks, stems, or mixtures thereof, are preferably used. Preferably, the leaves or seeds of the acid garlic are used. Because, the leaves and seeds of mountain garlic can be easily obtained at any time, and can be easily obtained without requiring a separate process when obtaining the extract.

In addition, the present invention provides an anti-inflammatory composition comprising an acid garlic extract as an active ingredient.

The anti-inflammatory composition includes a pharmaceutical composition and a functional food comprising an acid garlic extract as an active ingredient.

The acid garlic extract included in the pharmaceutical composition may be an extract obtained from seeds, leaves, roots, flower stalks and stems of acid garlic, and may be a mixture thereof.

The pharmaceutical composition may be used for the prevention or treatment of rheumatoid arthritis or atherosclerosis. Human umbilical vein endothelial cells (HUVECs) damaged by free radicals cause adsorption of neutrophiles and monocytes in the joint and act as a cause of aggravating rheumatoid arthritis . In addition, the adsorption reaction between mononuclear leukocytes and HUVEC cells occurs in the early stages of the immunological inflammatory response. The protein involved in cellular adhesion molecule (CAM) is mediated by the immunological inflammatory mediator TNF (tumor necrosis factor) -α. Increased expression and increased expression of CAM promotes the adsorption of mononuclear leukocytes and neutrophils, leading to rheumatoid arthritis and atherosclerosis. However, the acid garlic extract according to the present invention has the effect of inhibiting the intercellular adsorption of HUVECs and mononuclear leukocytes and inhibiting the expression of CAM, as shown in the following experimental example, preventing or treating rheumatoid arthritis or atherosclerosis. Useful for

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 during actual clinical administration, and when formulated, diluents such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc. that are commonly used, or It is prepared using excipients. 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, Uittepsol, macrogol, Tween 61, cacao butter, laurin butter, glycerol gelatin and the like can be used.

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

In addition, the present invention provides a functional food comprising an acid garlic extract as an active ingredient, the functional food is used for the prevention of rheumatoid arthritis or atherosclerosis due to the effect of the acid garlic extract.

Acid garlic extract according to the present invention has an antioxidant effect, in particular, the extract of the leaf part of the acid garlic has a relatively high antioxidant effect compared to other parts of the acid garlic. In addition, the acid garlic extract according to the present invention had the effect of protecting HUVECs from superoxide anions secreted from activated neutrophils. In addition, there was an effect of inhibiting the intercellular adsorption of mononuclear leukocyte cells and HUVEC monolayer, in particular, the extract of the garlic seed site was effective to inhibit the intercellular adsorption to the basic level. In addition, the acid garlic extract of the present invention has an effect of inhibiting the expression of CAM protein that is increased by TNF-α, an immunological inflammatory mediator, and develops rheumatoid arthritis and atherosclerosis. Accordingly, the acid garlic extract of the present invention can be used for the prevention or treatment of rheumatoid arthritis or atherosclerosis by inhibiting the adsorption of HUVECs and mononuclear leukocytes, which act as mediators of inflammatory diseases, and inhibiting the expression of related genes CAM.

Hereinafter, the present invention will be described in detail by the following examples. However, the present invention is not limited by the following examples.

Example  1: Preparation of Mountain Garlic Extract

Mountain garlic was separated into roots, seeds, leaves, flower stalks and stems, washed, and dried in vacuo to be crushed. To 30 g of the ground garlic powder, 100 ml of 70% ethanol was added and extracted at room temperature for 24 hours. The resulting mixture was filtered and the solvent was evaporated under reduced pressure and concentrated to give an acid garlic extract.

Experimental Example  1: Antioxidant Activity of Acid Garlic Extract

(1) DPPH radical scavenging activity

Using the same method as in Example 1, an extract of each part of the garlic was prepared from seeds, leaves, flower stalks, stems, and roots of the garlic, and the DPPH scavenging activity of the extract of the garlic was measured by a conventional method. To 0.2 ml of methanol solution (1,000 μg / ml) containing each of the garlic extracts, DPPH (1,1-diphenyl-2-picryl hydrazyl) methanol solution (1 mmol / ml, 0.5 ml) was added. The resulting mixture was stirred for 15 seconds and incubated for 30 minutes at room temperature. Absorbance was measured at 517 nm. As a control, a DPPH methanol solution containing no acid garlic extract was used. The results are shown in Table 1 below.

Table 1: DPPH Radical Scavenging Activity of Mountain Garlic Extract

Mountain garlic DPPH radical scavenging activity (%) ^{1 )} Seed 1.69 ± 0.72 leaf 51.39 ± 4.51 Flower bed ND ^{2 )} stem ND Root 5.29 ± 0.66

1) DPPH radical scavenging activity is expressed as a ratio with respect to the control

2) ND means no activity was detected.

As salping in Table 1, acid garlic extract showed DPPH radical scavenging activity. In particular, the DPPH radical scavenging activity of the leaf extract was 51.4%, which was superior to the extract of the other portions. In contrast, the stalk and stem did not show DPPH radical scavenging activity.

(2) reducing power

Using the same method as in Example 1, an extract of each part of the garlic was prepared from seeds, leaves, flower stalks, stems, and roots of the garlic. Each acid garlic extract was added to distilled water to make 1000 ㎍ / ml. The resulting solution was 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 . The resulting mixture was reacted at 50 ° C. for 20 minutes. 2.5 ml of 10% trichloroacetic acid was then added and centrifuged for 10 minutes at 2,090 × g. 2.5 ml of the supernatant in the resulting mixture was mixed with 2.5 ml of distilled water and 0.5 ml of 0.1% FeCl ₃ , and the absorbance was measured at 700 nm using a UV-spectrometer (Agilent Technologies Inc., Santa Clara, Calif., USA). The results are shown in Table 2.

Table 2: Reducing power of acid garlic extract (concentration of acid garlic extract is 1,000 ㎍ / ml)

Mountain garlic Reducing power Seed 0.11 ± 0.00 leaf 0.42 ± 0.01 Flower bed 0.27 ± 0.01 stem 0.09 ± 0.00 Root 0.05 ± 0.00

As salping in Table 2, it showed the reducing power of the acid garlic extract. In particular, the leaf extract showed a much higher reducing power than the extract of the other parts. In addition, the extracts of the peduncle and leaf showed higher reducing power than the extracts of root and stem.

Experimental Example  2: of mountain garlic extract Neutrophil  From leukocytes HUVEC  Protective effect

(1) Isolation of Neutrophiles (PMN)

Whole blood was taken from healthy adult men and treated with heparin (Sigma-Aldrich Inc., (St. Louis, MO, USA)) to prevent clotting. Histopaque-1083 (Sigma-Aldrich Inc., (St. Louis, Mo., USA)) was added and centrifuged to separate neutrophil leukocytes from other intracellular particles. Neutrophil leukocytes were then isolated by precipitation with dextran solution (dextran T-500, Amersham Biosciences Inc., Uppsala, Sweden) and osmotic pressure difference using hypotonic saline. The isolated neutrophils were 1 x 10 ⁷ cells / ml using Han's balanced salt solution (HBSS) without calcium, magnesium and phenol red (GIBCO, Invitrogen Inc., Grand Island, NY, USA) (pH 7.4). It was suspended and used at a concentration of.

(2) HUVEC protection effect from activated neutrophils

To a certain amount of neutrophils prepared above was added fMLP (n-formyl-methionyl-leusil-phenylalanine) (Sigma-Aldrich Inc., (St. Louis, MO, USA)) (1 uM) and 37 hours The secretion of superoxide from neutrophils was activated by culturing in a 5% CO ₂ humidified incubator. fMLP is a biochemical that stimulates neutrophils to secrete superoxide. Human umbilical vein endothelial cells, HUVEC (CRL-2480, ATCC, Manassas, VA, USA) were seeded in 96 well plates at 1 × 10 ⁵ concentration and incubated for 24 hours. The cultured HUVECs were then incubated in a 5% CO ₂ humidified incubator for 2 hours by adding 1 × 10 ⁵ concentration (100 μl) of neutrophils activated with fMLP. The degree of cytotoxicity to HUVECs was then assessed by conventional MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) method. Specifically, MTT (Sigma Aldrich Inc., (St. Louis, MO, USA)) solution was added so that each well was at a final concentration of 0.5 mg / ml and incubated at 37 ° C. for 4 hours. All medium was removed from the plate and 100 μl of DMSO was added to each well to allow the resulting formazan to elute at room temperature for 5 minutes. Absorbance was measured at 540 nm using a UV-spectral plate reader (Emax, Molecular Devices Inc., Sunnyvale, Calif., USA). As a control, absorbance was measured in the same manner by adding neutrophils which were not treated with fMLP.

Survival of HUVECs is expressed as the ratio to HUVEC monolayers in the fMLP-treated group. The results are shown in FIG.

As shown in Figure 1, the HUVEC treated with the garlic extract of the present invention showed a nearly similar HUVEC survival rate as the group not treated with fMLP. Therefore, it can be seen that the acid garlic extract of the present invention has an effect of protecting HUVEC from superoxide anions secreted from activated neutrophils. Specifically, the seed part extract of acid garlic showed 94% protection of HUVEC, and the extract of leaf part showed 85% protection effect. In addition, this protective effect was increased with increasing the concentration of the acid garlic extract.

Experimental Example  3: Cytotoxicity of Acid Garlic Extract cytotoxicity ) Research

(1) cell culture

(a) HUVEC culture

Human Umbilical Vein Endothelial Cells (HUVEC) (CRL-2480, ATCC, Manassas, VA, USA) were prepared using 10% FBS, penicillin / streptomycin 100 units / ml, heparin 0.1 mg / ml, and endothelial cell growth supplement (GIBCO, Invitrogen). Inc., Grand Island, NY, USA) was cultured in a 37 ° C., 5% CO ₂ humidified incubator using a F-12K nutrition mixture (Kaighn's modification, GIBCO, Invitrogen Inc.,) containing 0.03 mg / ml. The HUVEC monolayer was washed twice with PBS (pH 7.4) to remove serum components containing trypsin inhibitor in the medium, followed by 0.05% trypsin (0.53 mM EDTA) (Gibci, Invitrogen Inc., Grand Island, NY, USA) was used to passage the cells.

(b) Culture of Rat Liver Epitherial Cells

Rat liver epithelial cells (WB-F344 cell, East Lansing, MI, USA) at 37 ° C, 5 using DMEM (Gibco, Invitrogen Inc.,) medium containing 10% FBS and 100 units / ml of penicillin / streptomycin Cultured in a% CO ₂ incubator. Passage was used with 0.05% trypsin.

(2) cytotoxicity investigation

WB-F344 cells and HUVECs were incubated in a 37 ° C., 5% CO ₂ humidified incubator for 24 hours at a concentration of 1 × 10 ⁴ cells / well using 96 well culture plates (Corning Inc., Corning, NY, USA). . Using the seeds, leaves, and roots of mountain garlic, an extract of each part of mountain garlic was prepared in the same manner as in Example 1. Acid garlic extract was added and incubated for 24 hours. For HUVEC, the garlic extract was used at concentrations of 50 μg / ml, 100 μg / ml, 250 μg / ml, 500 μg / ml and 1000 μg / ml. For the WB-F344 cells, acid garlic extract was used at concentrations of 3.125 μg / ml, 6.25 μg / ml, 12.5 μg / ml, 25 μg / ml, 50 μg / ml, and 100 μg / ml. MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) (Sigma Aldrich Inc., (St. Louis, MO, USA)) solution was added to each well. A final concentration of 0.5 mg / ml was added and incubated at 37 ° C. for 4 hours. All medium was removed from the plate and 100 μl of DMSO was added to each well to allow the resulting formazan to elute at room temperature for 5 minutes. Absorbance was measured at 540 nm using a UV-spectral plate reader (Emax, Molecular Devices Inc., Sunnyvale, Calif., USA). As a control, a group of cells not treated with acid garlic extract was used. Survival was defined as the absorbance ratio of the wild garlic extract treated group to the control group. The results are shown in Tables 3 and 4 below.

Table 3: Cytotoxicity of Mountain Garlic Extract against HUVEC

Acid Garlic Extract Concentration Mountain garlic extract site Seed leaf Root 0 100.4 ± 5.7 100.4 ± 5.7 100.4 ± 5.7 50 95.6 ± 4.8 98.8 ± 11.6 105.9 ± 4.2 100 93.8 ± 7.5 92.3 ± 7.9 106.5 ± 4.7 250 100 ± 6.8 104.8 ± 6.9 117.4 ± 1.3 500 107.2 ± 2.5 126.9 ± 4.2 139.2 ± 6.1 1000 122.0 ± 5.4 181.3 ± 9.6 132.5 ± 8.0

Table 4: Cytotoxicity of Mountain Garlic Extracts against WB-F344 Cells

Acid Garlic Extract Concentration Mountain garlic extract site Seed leaf Root 0 100 ± 2.2 100 ± 5.4 100 ± 2.2 3.125 85.3 ± 2.1 86.3 ± 3.2 80.1 ± 8.9 6.25 80.2 ± 5.5 80.0 ± 5.3 79.2 ± 3.5 12.5 78.1 ± 3.3 82.2 ± 4.9 70.7 ± 6.3 25 72.5 ± 1.1 68.2 ± 4.2 74.8 ± 5.4 50 60.1 ± 3.1 74.5 ± 4.6 70.1 ± 3.7 100 57.2 ± 2.2 77.6 ± 7.8 66.5 ± 6.0

As salping in Tables 3 and 4 above, acid garlic extract did not show cytotoxicity against HUVEC monolayers. However, WB-F344 cells showed cytotoxicity. Accordingly, it can be seen that the acid garlic extract showed severe cytotoxicity in WB-F344 cells, but did not show cytotoxicity specifically for cell proliferation of HUVEC monolayers related to vascular dysfunction. Thus, cell adsorption experiments of acid garlic extracts were performed on HUVEC monolayers.

Experimental Example  4: Of Garlic Extract Mononuclear  Leukocyte cells and HUVEC Adsorption inhibition effect

(1) cell culture

(a) THP-1 Cell Culture as Monocytic Cells

THP-1, a mononuclear leukocyte cell, was purchased from the Korean Cell Line Bank (KCLB). Mononuclear leukocytes were cultured in 37 ° C., 5% CO ₂ incubator in RPMI-640 (GIBCO, Invitrogen Inc., Grand Island, NY, USA) medium containing 10% FBS and 100 units / ml of penicillin / streptomycin. . Cells were harvested by centrifugation of the cultured cell suspension at 2,090 × g for 2 minutes and passaged.

(b) HUVEC culture

Human umbilical vein endothelial cells (HUVEC) (CRL-2480, ATCC, Manassas, VA, USA) were added with 10% FBS, penicillin / streptomycin 100 units / ml, heparin 0.1 mg / ml and ECGC 0.03 mg Incubated in a 37 ° C., 5% CO ₂ humidified incubator using F-12K nutritional mixture (Kaighn's modification, GIBCO, Invitrogen Inc.,) containing / ml. The HUVEC monolayer was washed twice with PBS (pH 7.4) to remove serum components containing trypsin inhibitor. Cells were passaged using 0.05% trypsin (0.53 mM EDTA).

(2) cell adsorption assay

Subcultured mononuclear leukocytes (THP-1 cells) were treated with 5 μM calcein-AM (calcein O, O'-diacetate tetrakis (acetoxymethyl) ester) (Sigma-Aldrich Inc., (St. Louis, MO) , USA) (PBS solution, pH 7.4) for 30 minutes at 37 ° C. to label mononuclear leukocytes with fluorescein calcein-AM, then wash three times with PBS to label cells and the remaining calcein-AM To perform adsorption experiments, labeled cells were suspended and used in RPMI-640.

HUVECs passaged for cell adsorption analysis were seeded at a density of 1 × 10 ⁵ cells / well in 96 well tissue culture plates (Corning 3603, Corning In.). After incubation at 37 ° C. for 24 hours, the cultivation was continued for 24 hours by adding acid garlic extract (1000 μg / ml) to the obtained HUVEC single layer. Acid garlic extract was obtained using the method of Example 1 for seeds, leaves or roots of mountain garlic. Then 5 ng / ml of TNF-α (BD Science, San Jose, Calif., USA) was added to activate cell adsorption for 24 hours. Washed three times with PBS before cell adsorption experiment.

THP-1 labeled with calcein-AM was mixed with the activated HUVEC at a concentration of 5 x 10 ⁵ cells / well and incubated in a 37 ° C., 5% CO ₂ humidified incubator for 1 hour. Washed four times with PBS to remove unsorbed THP-1. THP-1 labeled with calcein-AM adsorbed on HUVEC was measured using a fluorescence plate reader (FL600, Bio-Tek Instruments, Inc., Winooski, VT, USA). The excitation / luminescence wavelength of calcein-AM measured at this time was 485 nm / 530 nm. The results are shown in Table 5 below.

Table 5: Effect of HUVEC Monolayer and THP-1 Inhibition on Cell Adsorption

Wild vegetable extraction site Inhibitory Effect (%) Seed 105.75 leaf ND ^{1 )} Root 45.74

1) ND: no inhibitory effect detected

As shown in Table 5, the acid garlic extract of the present invention showed a cell adsorption inhibitory effect of HUVEC monolayer and THP-1. In particular, extracts from wild garlic seeds inhibited cellular adsorption of HUVEC monolayers and THP-1 to basal levels.

In order to further confirm the effect of inhibiting cell adsorption, HUVECs were cultured in 24-well culture plates, and adsorption with calcein-AM-labeled THP-1 was inverted fluorescence microscope (IX 71). , OLYMPUS iNC., Tokyo, Japan) at 100 and 200 magnification. The used garlic extract is an extract obtained from the root of the garlic. Observed in real time using an OLYMPUS DP50 camera attached to a microscope (Imaging software, ViewfinderLite, 1.0.134 version, Pixera Corporation, Los GATOS, USA, OLYSIA BioAutoCell 3.2 version, SoftImaging System, Tokyo, Japan) . The photosynthesis of TNF-α-activated and basal level photos were used to determine the inhibitory effect of cell roots on the adsorption of wild roots. The results are shown in FIG.

As shown in Figure 2, it can be seen that the extract of the garlic seed site of the present invention inhibited the cell adsorption of HUVEC monolayer and THP-1 to a basal level.

Experimental Example  5: Of Garlic Extract CAM  Protein Transcription Electrolytic Effect

The transcriptional inhibitory effect of acid garlic extract on vascular cellular adhesion molecule-1 (VCAM-1), intracellular adhesion molecule-1 (IMAM-1), and E-selectin was examined.

For the seed, leaves and roots of mountain garlic, an extract of each part of the mountain garlic was prepared in the same manner as in Example 1. Mountain garlic extract was preincubated for 30 minutes by adding 0.5 mg / ml, 1 mg / ml, 5 mg / ml to the HUVEC single layer. Then, 10 ng / ml of TNF-α was added and incubated for 6 hours. RNA was extracted from HUVEC using RNeasy kt (Quiagen Inc., Valencia, CA, USA). RT-PCR was performed using a One-Step RT-PCR kit from Quiagen and Bioneer. Primers used for PCR are shown in Table 6 below, and the final concentration was 1 μM.

Table 6: Primers for RT-PCR (FP: forward primer, RP: reverse primer)

gene Primer sequence VCAM-1 (FP) 5'-ATGCCTGGGAAGATGGTCGTGA-3 ' VCAM-1 (RP) 5'-TGGAGCTGGTAGACCCTCGCTG-3 ' 1CAM-1 (FP) 5'-GGTGACGCTGAATGGGGTTCC-3 ' ICAM-1 (RP) 5'-GTCCTCATGGTCGGGCTATGACTC-3 ' E-selectin (FP) 5'-ATCATCCTGCAACTTCACC-3 ' E-selectin (RP) 5'-ACACCTCACCAAACCCTTC-3 ' GAPDH (FP) 5'-ATGACAACAGCCTCAAGATCATCAG-3 ' GAPDH (RP) 5'-CTGGTGGTCCAGGGGTCTTACTCCT-3 '

For comparison of transcriptional efficiency, the transcriptional efficiency of GAPDH gene was compared. PCR was performed using a Bio-RAD thermal cycler (MJ Mini, Bio-Rad Inc., Hercules, Calif., USA), RT was 30 minutes at 50 ° C. for the synthesis and predenaturation of cDNA. , 15 minutes at 95 ° C. PCR amplification was carried out 30 times for 1 minute (denatured) at 95 ° C., 2 minutes (annealed) at 55 ° C., and 3 minutes (extended) at 72 ° C. Finally, the final expansion for 10 minutes at 72 ℃. The RT-PCR product obtained was stored at 4 ° C. until separated on agarose gel. As a control group, a group not treated with TNF-α and a group not treated with TNF-α and not treated with garlic extract were used. The result of separation in the agarose gel is shown in FIG. 3.

As shown in Figure 3, when TNF-α was not treated, no band was detected in the basic mRNA transcription result of the CAM protein in HUVEC. However, when treated with TNF-α, transcription of the CAM protein increased significantly. When the acid garlic extract of the present invention was treated, mRNA transcription of CAM was inhibited. The extracts from the garlic seed area had a slight effect on the transcription of mRNA in the case of ICAM-1, while significantly reducing the transcription of VCAM-1 and E-selectin. Inhibited. In addition, it can be seen that the extracts of the leaves and roots of wild garlic inhibited the degree of transcription of VCAM-1.

Figure 1 shows the effect of acid garlic extract (leaf, seed, root extract of garlic) to protect HUVEC against activated neutrophils.

Figure 2 shows the inhibitory effect of the seed extract of acid garlic on the cell adsorption of THP-1 and HUVEC single layer in fluorescence images at 100 magnification (A) and 200 magnification (B).

Figure 3 shows the inhibitory effect of acid garlic extract (seed garlic seed, leaf, root extract) on the transcriptional expression of CAM mRNA in HUVEC activated with TNF-α.

Claims (8)

Mountain Garlic ( Allium victorialis subsp . platyphyllum ) antioxidant acid garlic extract. Mountain Garlic ( Allium victorialis subsp . platyphyllum ) anti-inflammatory acid garlic extract. The acid garlic extract according to claim 1 or 2, wherein the acid garlic extract is extracted by adding one or more solvents selected from the group consisting of ethanol, methanol, propanol, chloroform and acetone. The extract of claim 1, wherein the extract of the garlic is extracted from the leaves or seeds of the garlic. A pharmaceutical composition for preventing or treating an anti-inflammatory disease, comprising the extract of claim 1 or 2 as an active ingredient. 6. The composition of claim 5, wherein the anti-inflammatory disease is rheumatoid arthritis or atherosclerosis. Functional food for the prevention of anti-inflammatory diseases, comprising the acid garlic extract of claim 1 or 2 as an active ingredient. The functional food according to claim 7, wherein the anti-inflammatory disease is rheumatoid arthritis or atherosclerosis.

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