KR100454087B1 - Tilianin for anti-atherogenic activity - Google Patents

Abstract

The present invention relates to tilianin that exhibits anti-arteriosclerosis activity, and more particularly, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion-1, which are inflammatory response factors. It is excellent in anti-inflammatory activity such as suppressing the expression of molecule-1, VCAM-1), and it has an effect of remarkably reducing the development of atherosclerotic lesions based on the inflammatory response, which is useful for preventing and treating atherosclerosis. It's about (tilianin).

Description

Tilianin for anti-atherogenic activity

The present invention relates to tilianin that exhibits anti-arteriosclerosis activity, and more particularly, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion-1, which are inflammatory response factors. It is excellent in anti-inflammatory activity such as suppressing the expression of molecule-1, VCAM-1), and it has an effect of remarkably reducing the development of atherosclerotic lesions based on the inflammatory response, which is useful for preventing and treating atherosclerosis. It's about (tilianin).

Inflammatory reactions involve damage to tissues (cells) or external infectious agents (bacteria, fungi, viruses, various types of allergens), which involve various enzymes and immune cells in local blood vessels and body fluids. A series of complex physiological reactions, including substance secretion, fluid infiltration, cell migration and tissue destruction, and external symptoms such as erythema and edema fever pain. In normal cases, inflammatory reactions restore the function of life by removing external infectious agents and regenerating damaged tissues.However, if the inflammatory response is excessive or persistent due to the absence of antigens or internal substances, the major pathology of the disease (sensitizing disease) , Chronic inflammation) and also become a disorder in the treatment process such as blood transfusion, drug administration, and organ transplantation.

In connection with the present invention describes the action of the main factors of the inflammatory response and the developmental process of atherosclerotic lesions by them as follows.

ICAM-1 is a representative protein of the cell adhesion substance expressed on the surface of endothelial cells. In normal endothelial cells, they are expressed at very low levels, but when stimulated by cytokine-like inflammatory mediators such as TNF-α, inteferon-γ, and interleukin-1β, expression levels rapidly increase and inflammatory cells such as mononuclear cells and lymphocytes move in the bloodstream And inflammatory cells migrate to inflammatory tissues [Wegner CD et al, Science, 247 (1941), 456-459, 1990; Dustin, M. L. et al, J. Immunol. 137 (1) 245-254, 1986]. Therefore, ICAM-1 expression plays an important role in the amplification of the inflammatory response by acting at the early stage when the inflammatory cells migrate and focus on the site of inflammation.

VCAM-1 is a group of cell-adhesive substances expressed on the surface of vascular endothelial cells. When the atherosclerotic lesions are produced in apolipoprotein E-deficient mice (ApoE-/-), which are similar to the development of arteriosclerosis in humans, The expression level in endothelial cells is rapidly increased, and the increase in expression level of VCAM-1 is directly related to the concentration of cholesterol-low density lipoprotein (cholesterol-LDL) in plasma [Artherioscler. Thromb. Vasc. Biol. 18 (1998), 842-851. Therefore, VCAM-1 expressed in vascular endothelial cells acts to attach monocytes and lymph cells in blood when atherosclerotic lesions are induced, and these inflammatory cells focus under vascular endothelial cells to induce an inflammatory response. The lesion plays an important role in the development.

Arteriosclerosis is a disease in which the arteries are hardened by genetic factors related to lipid metabolism, environmental factors such as eating habits, and lack of smoking, which causes circulatory diseases such as heart disease and cerebrovascular disease. The hypothesis of the early onset of atherosclerosis is "responce-to-injury hypothesis," which causes vascular endothelial cells due to genetic variations, peroxides, hypertension, diabetes, elevated plasma homocysteine levels, and microbial infections. Is a dysfunction that does not maintain normal homeostasis. When endothelial cells become dysfunctional, cell adhesion substances are greatly expressed and cell permeability is increased, and adhesion of immune cells, platelets, lipids, etc. and permeability to tissues are increased, and these immune cells secrete inflammatory mediators and growth factors. Atherosclerotic lesions develop and develop due to back inflammatory reactions [Russel R., New England J. of Med. 340 (2), 115-126, 1999]. At this time, low-density lipoprotein (LDL) in the blood produces modified-LDL (MLDL) as a cause of oxidation, glycemic binding, integration, glycoprotein binding, etc., and they stimulate vascular endothelial cells and smooth muscle. And damage [Steinberg D., J. Biol. Chem. 272, 20963-20966, 1997; Griendling K.K. et al., Circulation 96, 3264-3265, 1997; Bavab M. et al, Artheriosler. Thromb. Vasc. Biol. 16, 831-42, 1996. Because of this, when VCAM-1 expression of endothelial cells and inflammatory mediator release of inflammatory cells are promoted, LDL enters and accumulates under the endothelial cells, and the accumulated LDL and oxidized MLDL are again immune cells such as macrophages and T lymphocytes. The process of inducing the influx and activation of the cancer is repeated to promote the inflammatory response of the lesion [Rajavashisth TB et al., Nature, 344, 254-257, 1990; Quinn M. T. et al., Proc. Natl. Acad. Sci. USA 84, 2995-2998, 1987. Thereafter, the lesions are necrotic by the action of hydrolase, inflammation mediator, growth factor, etc. released from the macrophages or lymphocytes introduced into the lesion, and monocytes are introduced into the necrotic lesions, smooth muscle migration and differentiation, and fibrous Through repetitive processes such as tissue formation, the lesion tissue develops into a complex fibrous lesion covered with fibers in the necrotic tissue with the MLDL nucleus [Fuster V. et al. eds. Artherosclerosis and coronary artery disease. vol. 1, 539-555; 585-594; vol. 2, 492-510, Philadelphia, Lippincott-Raven, 1996], the formation of thrombi from developed lesion tissue and the hardening of the arteries resulting in circulatory disorders such as impaired blood flow [Russel R., New England J. of Med. 340 (2), 115-126, 1999; Wong, M.-L. et al, Proc. Natl. Acad. Sci. USA 97 (15), 8681-8686, 2000].

Therefore, atherosclerosis occurs when the lipid content such as cholesterol and LDL in the blood is high, but it is not caused by simple fat accumulation, but the inflow and accumulation of fat below the arterial endothelial cells and the development of the lesions and A series of processes leading to cell necrosis are typical inflammatory reactions involving endothelial cells, macrophages and lymphocytes.

On the other hand, tilianin is a glucose glycoside compound of the flavonoid acecetin, which has been reported in various plants [Saengyak Hakhoechi, 31 (2000), 105-108; Yakhak Hoechi, 43 (2000), 143-149; Zhonggou Zhongyao Zazhi 24 (1999), 158-160; Chin. Pharm. J. (Taipei) 49 (1997), 13-20; Biochem. Syst. Ecol. 25 (1997), 181; Yaoxue Xuebao 26 (1991), 906-910; Rastit. Resur. 27 (1991), 50-54; Pharm. Acta Helv. 63 (1988), 206-208; Phytochemistry 24 (1985), 201-203; Pol. Acta Soc. Bot. Pol. 51 (1982), 403-411; Chem. Pharm. Bull. 29 (1981), 1777-1779; Rev. Lationoam. Quim. 12 (1981), 37-38; Pol. Acta Soc. Bot. Pol. Acta Soc. Bot. Pol. 49 (1980), 281-296; Rev. Lationoam. Quim. 10 (1979), 136; Farmatsiya (Moscow) 27 (1978), 24-28; Agric. Biol. Chem. 42 (1978), 475-477; Phytochemistry 14 (1975), 1443-1446; Khim. Prir. Soedin. 10 (1974), 96-97; Fenol'nye Soedin. Ikh Biol. Funkts., Mater. Vses. Simp., 1st (1968), volume date 1966, 85-89]. On the other hand, regarding the physiological activity of tilianin, antioxidant activity [J. Food Sci. Nutr. 4 (1999), 221-225; Indian J. Chem., Sect. B: Org. Chem. Incl. Med. Chem 36B (1997), 1201-1203], and xanthine oxidase inhibitory activity was investigated, but no activity has been reported [J. Nat. Prod. 51 (1988), 345-348.

However, studies on the anti-arteriosclerosis activity of tilianin have not been reported at home and abroad.

Accordingly, an object of the present invention is to provide tilianin, which has anti-inflammatory activity and anti-arteriosclerosis activity, which is useful as an inflammatory disease, atherosclerosis associated with an inflammatory response, and thereby preventing and treating circulatory diseases.

Figure 1 is treated with 10 or 100 μM of tilianin for 2 hours in human umbilical vein endothelial cells (HUVECs) and then treated with TNF-α (10 ng / ㎖) for 16 hours and VCAM-1 expression was measured by flow cytometry As shown, the degree of inhibition of VCAM-1 expression by tirianin is shown. Figure 2 is a photograph showing the atherosclerotic lesions of the cross section of the cardiac aorta after 8 weeks for the tilianin-free group (the lesion tissue under vascular endothelial cells: dark staining site).

Figure 3 is a photograph showing the atherosclerotic lesions of the cross section of the cardiac aorta after 8 weeks for the 1% tilianin treatment group (x lesions under vascular endothelial cells: dark staining sites).

The present invention is characterized by a composition having an anti-arteriosclerosis activity containing tilianin represented by the following formula (1) as an active ingredient.

Referring to the present invention in more detail as follows.

Tilianin according to the present invention has anti-arteriosclerosis activities such as ICAM-1 expression inhibitory activity and VCAM-1 expression inhibitory activity, and thus is very useful for the prevention and treatment of atherosclerosis by significantly reducing the development of atherosclerotic lesions.

Therefore, the present invention includes a drug or a food additive comprising tilianin as an active ingredient.

Tilianin according to the invention can be chemically synthesized or isolated from plants. For example, when obtained from a plant, it is extracted from lower ethanol with lower alcohol and subjected to silica gel column chromatography, followed by pure separation using high performance liquid chromatography or recrystallization method. The anti-inflammatory activity such as ICAM-1 expression inhibitory activity, VCAM-1 expression inhibitory activity, and inhibitory activity of atherosclerotic lesions of the tilianin were investigated.

Monocyte THP-1 cell line (THP) induced the expression of ICAM-1 with tumor necrosis factor-α (TNF-α). It strongly inhibits ICAM-1 expression on -1 monocytic leukemic cells (THP-1 cells), and VCAM-1 expression on human umbilical vein endothelial cells (HUVECs) shows strong inhibitory activity. In addition, at 1% feed to LDLR-/-mice, the atherosclerotic lesions in the aortic sinus were significantly reduced.

On the other hand, the present invention includes a drug or food additive containing tilianin as an active ingredient, the manufacturing method of the tilianin to the drug or food additive according to the known method.

When manufactured as a pharmaceutical, tilianin may be used by itself, but a formulation such as powder, granule, capsule, or injection may be prepared by mixing a pharmaceutically acceptable carrier, forming agent, diluent, and the like. It can be prepared and used as. In addition, the dosage level of tilianin according to the present invention may vary depending on body absorption, weight, age of the patient, sex, health diet, administration time, administration method, excretion rate, severity of disease, and the like. Tilianin is preferably administered at about 0.1 to 10 mg / kg body weight. Therefore, the composition containing the active ingredient of the present invention is to be prepared in consideration of the effective amount range, and the unit dosage form formulated in this way according to the judgment of the expert and the needs of the individual to monitor or observe the administration of the drug as needed Specialized medications can be used or administered at regular intervals.

On the other hand, when manufacturing as a food additive may be prepared by including tilianin according to the present invention in food materials such as beverages, teas, spices, gum, confectionery.

Medicines and food additives containing tilianin as an active ingredient described above are excellent in the treatment as well as the prevention effect of inflammatory diseases and atherosclerosis and circulatory diseases.

Hereinafter, the present invention will be described in more detail based on the following examples, but the present invention is not limited to the examples.

Example 1: Isolation and Purification of Tilianin

After harvesting and cutting the ground parts of the cultivated cultivated cultivated cultivars (Agastache rugosa, Labiatae), 120 kg of the dried samples were added with 120 L of methanol and extracted by standing for 3 days. The extract obtained by repeating this process three times was concentrated. To 3.5 kg of the extract. 2.5 kg of the extract was suspended in 10 L of water, and then 10 L of chloroform (chloroform) were added to separate the chloroform layer, and this was repeated twice to remove the chloroform layer. Butanol (n-butanol) was fractionated in the same manner as described above, and the butanol layer was concentrated to obtain 450 g of a fraction. 150 g of butanol fraction was taken and adsorbed onto 1 kg of silica gel, which was then placed on top of a column (20 x 75 cm) packed with 4 kg of silica gel and mixed with chloroform-methanol (methanol content: 5% to 100%). Tilianin containing fractions were eluted. To 300 g of the remaining butanol fraction, 10 L of methanol was added, followed by standing to obtain a precipitate, which was washed several times with methanol to obtain a precipitate containing 90% or more of tilianin. Tilianin was purified purely using high performance liquid chromatography.

Example 2: ICAM-1 Expression Inhibitory Activity of Tilianin

The inhibitory activity of ICAM-1 expression of THP-1 cells was examined for tilianin. The detailed process of the experiment is as follows.

THP-1 cells were in fetal bovine serum (GibcoBRL 26140-079) in RPMI-1640 medium [RPMI-1640 (GibcoBRL 23400-021) 1.62%, sodium bicarbonate 0.2%, penicillin and streptomycin mixed antibiotics 1%]. Cultured in a carbon dioxide incubator (5% carbon dioxide, 95% relative humidity, 37 ° C., CO ₂ incubator) using a culture solution added with 10% FBS). The assay sample is dissolved in DMSO, diluted to 5% or less in phosphate buffered salin solution (PBS) and added to the reaction solution at 5% so that the dissolved DMSO concentration does not exceed 0.25% in the final reaction solution. It was. Dispense 200 μl of THP1 cells (2.5 × 10 ⁵ cells / ml) into a 96-well plate for each well, and incubate for 60 minutes in a CO ₂ incubator at 37 ° C by adding 10 μl of the sample solution prepared at a constant concentration. Thereafter, TNF-α (10 μl, final concentration of 10 ng / ml) was added to induce the expression of ICAM-1, and again incubated for 16 hours in a CO ₂ incubator. The reaction solution was fixed with 25 μl of glutaraldehyde buffer (glutaraldehyde 2.08% in PBS), the cells were fixed to the plate, washed with PBST (0.005% tween-20 in PBS) and 3% skim milk (non-specific binding). The site was blocked, washed again, and then the primary antibody (anti-human ICAM-1) and the secondary antibody (anti-mouse IgG peroxidase conjugate) were added sequentially, followed by OPD Peroxidase substrate (Sigma). 200 µl of P-9187), in 0.05 M phosphate-citrate buffer) was added, and after 5-10 minutes, 50 µl of 3M HCl was added to stop the reaction. Afterwards, the absorbance of the reaction solution was measured at 490 nm using a spectrophotometer to measure ICAM-1 expression rate of THP-1 cells by TNF- α , and the expression inhibition rate by the sample was expressed by the following equations (1) and (2). Calculated.

As shown in Table 1, the tilianin-treated group showed a little lower ICAM-1 expression inhibitory activity than the dexamethasone-treated group known as an inflammatory agent, but showed an excellent inhibitory activity, and dexamethasone had a strong effect as a steroid inflammatory agent. While steroid inflammatory agents commonly have side effects commonly associated with long-term use (inhibition of kidney function, increased susceptibility to infection, diabetes, muscle atrophy, growth inhibition, osteoporosis, etc.), tilianin according to the present invention has no side effects and is safe in use.

Example 3 Inhibitory Activity of Tilianin VCAM-1 Expression

The inhibitory activity of VCAM-1 expression on human umbilical vein endothelial cells (hereinafter referred to as HUVECs) was investigated. The detailed process of the experiment is as follows.

HUVECs cells were 100 U / mL penicillin (EBM-2, Clonetics CC-3156, MD, USA) in EGM-2 BulletKit medium [Kit which contains a 500 ml bottle of Endothelial Cell Basal Medium-2 (EBM-2, Clonetics CC-3156, MD, USA)]. penicillin) and 100 μg / ml of streptomycin were used and cultured in a carbon dioxide incubator (5% carbon dioxide, 95% relative humidity, 37 ° C. or less, CO ₂ incubator). The assay sample was dissolved in DMSO and the concentration of DMSO in which the sample was dissolved was no more than 0.1% in the final reaction solution. HUVECs cells were used in two 10 cm cell dish plates (2 × 10 ⁶ cells) for each group, and the medium was exchanged before the samples were processed. First, tilianin was pretreated for 2 hours at final concentrations of 100 μM and 10 μM. TNF- α was then treated at 10 ng / ml for each group to induce the expression of VCAM-1 for 16 hours. Cell collection was first performed by washing twice with PBS and then treated with trypsin (0.025%) for 5 minutes. The harvested cells were centrifuged in a 15 ml tube to discard supernatant, suspended cell precipitate with PBS, and washed again by centrifugation. After washing twice with PBS, the cells were suspended in 100 µl of PBS to which 0.5% BSA (Bovine Serum Albumin) was added, and then mouse anti-human monoclonal antibody (Rb 1/9; 1 µg / ml) was added. Added. Induce 30 min of binding to single antibody on ice, wash cells three times with cold PBS, goat F (ab ') 2 anti-mouse IgG (immunoglobulin G) diluted 1:25 (W / W) in PBS It was reacted for 40 minutes on ice with FITC (fluorescein isothiocyanate) conjugated with. These cells were fixed with 1% paraformaldehyde and measured for inhibition of VCAM-1 expression by tirianin using a flow cytometer (FACScan, Bio-Rad, USA) [Equation 3]. As a result, it was confirmed that VCAM-1 expression of HUVECs cells was strongly inhibited in the tilianin treated group [Table 2, FIG. 1].

Example 4 Atherosclerotic Lesion Inhibitory Activity of Tilianin

The inhibitory activity of tilianin against atherosclerotic lesions was investigated. The detailed process of the experiment is as follows.

(Step 1) Breeding test animals and experiment of animal administration

The experimental animals used were divided into two groups of 10 low-density lipoprotein receptor deficient mice 6-8 weeks old (average weight 16.8 g) females. One group consumed one high-fat pellet feed (containing 15% fat, 1.25% cholesterol and 0.5% sodium cholate), and the other group received a pellet feed containing 1% tilianin in the feed. During the experiment, solid feed and water were freely supplied.

(Step 2) Measurement of Atherosclerotic Lesions in Experimental Animals

Eight weeks after the start of the experiment, all mice underwent ocular blood collection. The hearts and arteries of the mice were perfused with phosphate buffer (PBS) for 10 minutes and then perforated with paraformaldehyde for 5 minutes. After this procedure, the heart and arteries were removed and soaked in 10% neutral formalin for 24 hours, followed by OCT medium [OCT medium, 10.24% polyvinyl alcohol, 4.26% polyethylene glycol, 80.5% nonreactive ingredient, w / w, Life Science International, England, UK] and frozen at -70 ° C. The frozen tissue sections were prepared using a slicer maintained at −20 ° C., and 6 sections of 9 μm were prepared from the artery with visible heart valves at the starting point, and then stained with dye red oil. Staining and counterstaining were performed with Harris hematocillin. The stained sections were measured using an image analyzer to measure the average lesion area of each animal group, and the lesion inhibition activity of the tilianin-treated group against lesion development in the untreated group was calculated by the following equation (4).

In the inhibitory effect of tilianin on atherosclerotic lesions, the experimental group fed the feed containing 1% tilianin showed 41.9% reduction in lesion size compared to the non-treated group [Table 3]. As a result of staining the cross-sectional tissue of the coronary artery, it was observed that the size of the lesion (necrotic tissue) due to the inflammatory response was significantly reduced in the 1% tilianin-added group compared to the non-treated group [FIGS. 2 and 3. ]. Therefore, it was confirmed that tilianin significantly reduced the development of atherosclerotic lesions during the development of atherosclerotic lesions due to the accumulation of immune cells such as macrophages and inflammation under the endothelial cells.

As shown in Table 3, the 1% lovastatin-treated group showed a higher activity of inhibiting atherosclerotic lesions than the 1% tilianin-treated group, while lovastatin had side effects of hepatotoxicity, whereas tilianin according to the present invention had side effects. It is safe to use. In addition, it was confirmed that the tilianin treatment group according to the present invention shows a very excellent effect compared to the untreated group.

Example 5: Preparation of Tablets

Tilanine 1 g

7 g of lactose

1.5 g of crystalline cellulose

0.5 g of magnesium stearate

Total amount 10 g

The tablets were prepared by direct tableting method after mixing the ingredients listed above finely. The total amount of each tablet was 100 mg, and the content of the active ingredient was 1 mg.

Example 6 Preparation of Powder

Tilanine 1 g

5 g of corn starch

4 g of carboxy cellulose

Total amount 10 g

A powder was prepared by crushing and mixing the ingredients listed above. 100 mg of powder was added to the 6 times hard capsule to prepare a capsule.

Example 7: Toxicity Test

Toxicity experiments were performed for tilianin according to the present invention as follows.

The tilianin was dissolved in dimethylsulfoxide (DMSO), diluted with water, and then administered to the mice (10 mice per group) at 1 g / kg and observed for 7 days, but no rats died.

Example 8 tilianin containing beverage composition

Ingredients used in tilianin-containing beverage compositions include plum extract (plum concentrate (solid amount 69 ^o Bx), manufacturer: Hadong Agricultural Cooperative), quince, donkey, health, Schisandra chinensis (Seoul Kyungdong Market), grape juice (Manufacturer: Comax international corp., Solid content 65 ^o Bx) and pear juice (manufacturer: Hanmi Spice Chemical Co., Ltd., solid content 69 ^o Bx) were prepared and purchased respectively.

First, the Chinese herbal medicine, Chinese Angelica, Angelica, Health, Schisandra chinensis, and Cinnamon, each added 10 wt. Times of water, and then hot water extracted at 100 ° C. for 30 minutes. This was allowed to stand at 4 ° C. for 24 hours, centrifuged at 10,000 × g for 10 minutes, and the supernatant was taken to obtain an herbal extract.

In addition, the plum extract was used to dilute the plum concentrate (69 ^o Bx) to 10 ^o Bx, grape juice (65 ^o Bx) and pear juice (69 ^o Bx) was used as the stock solution.

As tiyaninin-containing beverage composition, 0.1% tilianin, plum extract 0.2%, health extract 0.3%, donkey extract 0.3%, cinnamon extract 0.01%, pear juice 5.0%, high fructose 17% were used and diluted in water to 100 After adjusting to ㎖ and sterilized for 15 seconds at 95 ℃, cooled to prepare a drink-type beverage product.

As described above, tilianin according to the present invention inhibits the activation, production and expression of various inflammatory factors, exhibits anti-inflammatory activity in animal models and significantly inhibits the occurrence of atherosclerotic lesions based on anti-inflammatory activity. Therefore, it is useful as a medicine or a food additive for the prevention and treatment of inflammatory diseases, arteriosclerosis associated with inflammatory reactions and the resulting circulatory diseases.

Claims (11)

delete delete delete delete The anti-arteriosclerosis active composition characterized in that it contains the tilianin represented by the following formula (1) as an active ingredient to inhibit ICAM-1 and VCAM-1 expression. Formula 1 6. The composition according to claim 5, wherein the tilianin is a synthetic chemical or a natural substance isolated from Agastache rugosa . delete delete delete delete delete