KR101698207B1 - Composition comprising plant extracts for prevention or treatment of thrombosis diseases - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating thrombotic diseases caused by blood circulation disorder and blood circulation improvement, which comprises a complex herbal medicine extract comprising Angelica gigas and Gardenia or a complex herbal medicine extract comprising Angelica gigas Nakai, And a health functional food for preventing or ameliorating thrombotic diseases caused by circulatory disturbance and blood circulation improvement. The herbal extracts of the present invention, which are active ingredients of the pharmaceutical compositions or health functional foods of the present invention, inhibit the antioxidative effect, the thrombolytic effect, the inhibition of cell adhesion protein expression, and the expression of vasoactive inflammatory enzyme and inhibit the production of blood coagulation- , Myocardial infarction, cerebral hemorrhage, stroke and cerebral infarction.

Description

[0001] The present invention relates to a composition for preventing or treating thrombotic diseases,

The present invention relates to a pharmaceutical composition for preventing or treating thrombotic diseases caused by blood circulation disorder and blood circulation improvement, which comprises a complex herbal medicine extract as an active ingredient, a health functional food for preventing or improving thrombotic diseases due to blood circulation disorder and blood circulation improvement .

In recent years, circulatory diseases such as cerebral hemorrhage, heart disease, stroke, and cerebral infarction have been the leading causes of death in Korea as well as advanced countries. A variety of causes can block the blood vessels and cause the diseases. If the cardiovascular system is blocked, myocardial infarction may occur. If the blood vessels of the brain are blocked, a cerebral infarction may occur. In the case of cerebral infarction, the disease may continue to progress, resulting in a stroke and death. In the United States, stroke is the third most common cause of death in adults and is known to be the most important cause of mobility. The main cause of these diseases is known as thrombus. Thrombosis is recognized as a pathological phenomenon mediated by excessive platelet aggregation. In addition, this causes a disorder of blood circulation.

Humans have a vital system to prevent wound healing and loss of blood, which is controlled and balanced by a complex process called platelet and plasma clotting, fibrin degradation, and coagulation inhibition. However, when these equilibrium conditions are broken down by various factors, the blood flow is interfered, and thrombosis is generated, and the function of the tissue is impaired.

Thrombosis refers to the formation of lumps or blood clots inside the blood vessels, blocking the flow of blood. When blood vessels are injured, blood vessels contract and platelets adhere to the collagen fibers exposed to the wound, releasing serotonin, adenosine diphosphate (ADP) and thromboxane A2 do. ADP causes more platelets to cling, and thromboxane A2 acts to aggregate platelets, causing blood vessels to contract. Blood coagulation occurs after the primary hemostasis. The fibrin produced after hemostasis is dissolved by fibrinolysis. The tissue-type plasminogen activator (t-PA) and urokinase (UK) are plasminogen (plasminogen) To decompose fibrin. These blood coagulation and fibrinolysis are balanced in the normal case, but when this balance is broken, thrombosis occurs in which blood clots are deposited and blood circulation obstruction occurs in the blood vessels ( Nesheim M., Chest . , 2003 Sep ; 124: 33S-9S).

Currently, antiplatelet agents, anticoagulants, thrombolytic agents for treating thrombosis formed, and the like are used for prevention and treatment of thrombotic diseases. Aspirin, a typical antiplatelet agent, is known to cause side effects such as gastrointestinal bleeding and peptic ulcer, although it is effective. In addition, most of the drugs used as other anticoagulants are not able to be administered orally, and their selectivity to thrombosis is low, and thus they show various side effects such as hemolysis, immune reaction, fever, and allergy.

Therefore, it is urgently required to develop a therapeutic agent having high selectivity against thrombogenesis inhibition such as inhibition of platelet activation and thrombosis, dissolution activity of thrombus, etc. and minimizing side effects.

On the other hand, Angelica gigas Nakai (Lynx) is a perennial herb that belongs to the periwinkle, which refers to the roots of the perennial herb in Oriental medicine. E-deficiency treatment action, anemia, analgesia, tongue, and is used as a sickness and wives.

Dansam is a perennial herbaceous plant, and has a red-brown color on its roots, which has a tanthinone compound, a phenanthraquinone pigment. Anticancer effect, antioxidant effect, and anti-inflammatory effect are known as pharmacological actions so far known for Dansamp.

Gardeniae Fructus has long been used as a food coloring agent in the gardenia (Gardenia jasmoides Ellis) belonging to the Rubiaceae family or as a fruit of other plants. In oriental medicine, it is used as anti-inflammatory, diuretic, antipyretic, sedative, antibacterial, bile secretion, and hepatitis treatment.

A pharmaceutical composition for preventing or treating thrombotic disorders due to blood circulation disorder and blood circulation improvement, which comprises a complex herbal composition as an active ingredient, and a health functional food for preventing or improving thrombosis due to blood circulation disorder and blood circulation improvement.

The present invention relates to a pharmaceutical composition for preventing or treating thrombotic diseases caused by blood circulation disorder and blood circulation disorder, which comprises, as an active ingredient, a complex herbal medicine extract comprising Angelica gigas and Angelica gigas or Angelica japonica, Danshen ginseng, and Gardenia.

The present invention also relates to a health functional food for preventing or ameliorating a blood circulation disorder and a blood clotting disorder, which comprises a complex herbal medicine extract containing Angelica keiskei keis, Angelica japonica, Gardenia sp.

In the present invention, the herbal medicine extract comprises 45 to 75% by weight of Angelica gigas Nakai, 25 to 55% by weight of gardenia, 35 to 65% by weight of Angelica gigas, 15 to 45% by weight of ginseng and 5 to 35% .

In the present invention, the thrombotic disease is selected from the group consisting of myocardial infarction, cerebral hemorrhage, stroke, and cerebral infarction.

In the present invention, the herbal medicine extract has a thrombolytic activity, inhibits the expression of cell adhesion protein, and inhibits the expression of vascular inflammation inducing enzyme.

The herbal medicine extract according to the present invention has an antioxidative effect and a thrombolytic effect and inhibits expression of cell adhesion protein, inhibits the expression of vascular inflammatory enzyme and inhibits the production of blood coagulation-related factors. Therefore, it can be useful for prevention and treatment of thrombotic diseases such as myocardial infarction, cerebral hemorrhage, stroke and cerebral infarction.

Figure 1 shows the antioxidative effects of the extracts prepared in Examples 1-3.
Fig. 2 is an analysis of the polyphenol contents of the extracts prepared in Examples 1 to 3. Fig.
Fig. 3 shows the cytotoxicity of the extracts prepared in Examples 1 to 3. Fig.
Figure 4 shows the thrombolytic activity of the extracts prepared in Examples 2 and 3.
FIG. 5 shows the inhibition of the expression of cell adhesion protein (ICAM-1) in the extracts prepared in Examples 2 and 3 by immunocytochemical staining.
FIG. 6 shows the inhibition of cell adhesion protein (VCAM-1) expression of the extracts prepared in Examples 2 and 3 by immunocytochemical staining.
FIG. 7 shows the inhibition of the expression of vascular inflammation-inducing enzyme (COX-2) in the extracts prepared in Examples 2 and 3 by immunocytochemical staining.
FIG. 8 shows the inhibition of vascular inflammation-inducing enzyme (5-LO) expression of the extracts prepared in Examples 2 and 3 by immunocytochemical staining.
FIG. 9 shows Western blotting of the extracts prepared in Examples 2 and 3 to inhibit the expression of vascular inflammation-inducing enzyme (COX-2, 5-LO).
FIG. 10 shows the inhibition of vascular inflammation-inducing activity (COX-2) activity of the extracts prepared in Examples 2 and 3 by ELISA.
FIG. 11 shows the inhibition of ACE enzyme by the extracts prepared in Examples 2 and 3. FIG.
12 shows the degree of suppression of blood coagulation-related factor (Thromboxane B2) production to the extracts prepared in Examples 2 and 3. [

Hereinafter, the present invention will be described in detail.

The present invention relates to a pharmaceutical composition for preventing or treating a blood clotting disorder and a thrombotic disease caused by improvement of blood circulation, comprising a complex herbal medicine extract comprising Angelica gigasum and Angelica gigas or Angelica gigas To provide a health functional food for preventing or ameliorating thrombotic diseases.

In the present invention, a pharmaceutical composition for preventing or treating thrombotic diseases due to blood circulation disorder and blood circulation improvement, a complex herbal medicine extract which is an active ingredient of a health functional food for preventing or improving thrombotic diseases due to blood circulation disorder and blood circulation improvement, Can be obtained through the same two steps.

The present invention relates to a method for drying a ginger powder, a ginseng, and a gardenia by natural drying, freeze-drying after rapid freezing, drying by hot air drying or low-temperature drying, preferably at 20 to 100 캜 for 10 to 80 hours, more preferably at 40 캜 Dried at 50 to 80 ° C. for 20 to 60 hours and then added with water, alcohol, hexane or a mixed solvent thereof containing purified water of 1 to 40 times, preferably 2 to 20 times the weight of Angelica gigas Nakai, Using an extraction method such as hot water extraction, reflux circulation extraction, pressure extraction or ultrasonic extraction for 2 hours to 6 hours at 20 ° C to 100 ° C for 1 hour to 10 hours, preferably 40 ° C to 80 ° C, Filtering the extract; And the filtered extract is concentrated under reduced pressure or lyophilized and spray dried to obtain Angelica gigas Nakai, Dansamp and Gardenia extract.

It is preferable to use C1 to C4 lower alcohols as the solvent for extracting the Angelica gigasso, Danamia, and Gardenia extract, more preferably ethanol, methanol or butanol, more preferably 10 to 50% Ethanol (alcohol, alcohol) is used.

In addition, the extracts may be mixed to produce a complex herbal medicine extract. The herbal extract may be prepared by mixing a herbal medicine extract comprising 45 to 75% by weight of Angelica gigas Nakai and 25 to 55% by weight of gardenia, 35 to 65% 5 to 35% by weight of gardenia can be mixed to prepare a herbal medicine extract.

The herbal extracts obtained by the above methods have antioxidative and thrombolytic effects, inhibit the expression of cell adhesion proteins, inhibit the expression of vascular inflammatory enzymes and inhibit the production of blood coagulation factors. Therefore, the herbal medicine extract may be useful for the prevention and treatment of thrombotic diseases such as myocardial infarction, cerebral hemorrhage, stroke and cerebral infarction.

The pharmaceutical composition may be administered orally or parenterally, and it is preferable to employ an external or intraperitoneal injection, intramuscular injection, subcutaneous injection, intravenous injection, intramuscular injection, or intra-thoracic injection injection for parenteral administration .

The pharmaceutical composition may further contain conventionally used excipients, disintegrants, sweeteners, lubricants, flavors, and the like. Examples of the disintegrant include sodium starch glycolate, crospovidone, croscarmellose sodium, alginic acid, carboxymethylcellulose calcium, carboxymethylcellulose sodium, chitosan, guar gum, magnesium aluminum silicate, and polacrilin potassium.

In addition, the pharmaceutical composition may further include a pharmaceutically acceptable additive. Examples of the pharmaceutically acceptable additives include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate Starch glycolate, sodium starch glycolate, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, calcium stearate, white sugar, starch, sodium carboxymethylcellulose, Dextrose, sorbitol, talc, and the like may be used. The pharmaceutically acceptable additives according to the present invention are preferably contained in an amount of 0.1 to 90 parts by weight based on the pharmaceutical composition.

Solid form preparations for oral administration include powders, granules, tablets, capsules, soft capsules, and the like. Examples of liquid formulations for oral use include suspensions, solutions, emulsions, syrups and aerosols. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, fragrances and preservatives Examples of the agent for parenteral administration include external preparations such as powders, granules, tablets, capsules, sterilized aqueous solutions, liquid preparations, non-aqueous preparations, suspensions, emulsions, syrups, suppositories and aerosols, The pharmaceutical composition for external use for skin of the present invention may be formulated in the form of an injectable preparation and preferably used as a cream, gel, patch, spray, ointment, warning agent, lotion, liniment, pasta or cataplasma But is not limited thereto. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

The preferred dosage of the pharmaceutical composition may be appropriately selected by those skilled in the art depending on the degree of absorption, inactivation rate and excretion rate of the active ingredient in the body, age, sex and condition of the patient, and severity of the disease to be treated. For the desired effect, however, in the case of an oral administration, administration of the composition of the present invention at a daily dose of 0.0001 to 100 mg / kg, preferably 0.001 to 100 mg / kg per 1 kg of body weight per day to an adult good. The administration may be carried out once a day or divided into several times.

Further, the health functional food containing the complex herbal medicine extract of Angelica gigas Nakai and the gardenia of the present invention, or the herbal extract of Angelica gigas Nakai, ginseng root, and the gardenia as an active ingredient may be used together with other food or food ingredients, Can be suitably used. The amount of the active ingredient to be mixed can be suitably determined according to the intended use (prevention, health or hygiene).

There is no particular limitation on the form and kind of the health functional food. The form of the health functional food to which the substance can be added may be tablets, capsules, powders, granules, liquids, and rings. The types of health functional foods include dairy products including butter, yogurt, cheese, dairy products including ice cream, Such as lactic acid bacteria preparation, fermented milk, bread, chocolate, candy, snack, confectionery, pizza, ramen, other noodles, gum, various soups, drinks, tea, drink, alcoholic drinks and vitamin complex.

The health functional food of the present invention may contain various flavoring agents or natural carbohydrates as an additional ingredient such as a normal health functional food. The above-mentioned natural carbohydrates are sugar alcohols such as monosaccharides such as glucose and fructose, polysaccharides such as disaccharides such as maltose and sucrose, dextrin and cyclodextrin, and xylitol, sorbitol and erythritol. Examples of sweeteners include natural sweeteners such as tau martin and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like.

In addition to the above, the health functional food of the present invention may contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acids and salts thereof, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, Alcohols, carbonating agents used in carbonated drinks, and the like. These components may be used independently or in combination. The proportion of such additives is not critical, but is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the examples.

< Example  1> Angelica, Sansam , Manufacture of gardenia ethanol extract

(Ethanol, alcohol, alcohol, alcohol) was added, and the mixture was shaken at 60 ° C for 4 hours. The mixture was filtered, 30% ethanol extract of Dansam and Gardenia was prepared. After the extract was filtered, the filtrate was concentrated under reduced pressure at 55 ° C for 3 hours and lyophilized to use as an analytical sample.

< Example  2> Manufacture of complex herbal medicine extract of Angelica gigas and Gardenia

A mixed herbal extract (saccharide 6-value 4) was prepared by mixing 60% by weight of Angelica gigantosa extract and 40% by weight of gardenia extract obtained in Example 1 and used as an analytical sample.

< Example  3> Angelica, Sansam , Herbal extract of gardenia

A complex herbal extract (5-stearate 3-valent sugar 2) was prepared by mixing 50% by weight of Angelica gigantosa extract, 30% by weight of ginseng extract and 20% by weight of gardenia extract obtained in Example 1 and used as an analytical sample.

< Experimental Example  1> DPPH free Radical  Scavenging activity (Antioxidant effect test)

To the extracts obtained in Examples 1 to 3, 750 μl of DPPH (0.2 mM in ethanol) and 675 μl of tertiary purified water were mixed and reacted at room temperature for 30 minutes. 200 μl of the reaction solution was transferred to a 96-well plate, and the absorbance was measured at 520 nm using an ELISA reader. The DPPH radical scavenging activity (%) was calculated from the equation (1 - absorbance of the sample added sample / absorbance of the sample not added group) × 100, and the concentration of the sample (SC50) required to eliminate 50% of the generated radical was expressed in ppm Respectively. Experimental results were expressed as means ± standard error (mean ± SEM) by repeating 3 times and compared by statistical treatment with Student's t-test. Statistical significance was determined when the p value was less than 0.05 (p <0.05).

As shown in FIG. 1, the herbal extract (saccharide 6-type 4) obtained in Example 2 showed a high scavenging activity in the radical scavenging activity test using the DPPH reagent.

< Experimental Example  2> Polyphenol content analysis (antioxidant, cholesterol lowering, anti-cancer effect experiment)

To determine whether the extracts obtained in Examples 1 to 3 have various physiological activities such as antimutagenic, cholesterol-lowering, anticancer and antioxidative activities, polyphenol contents were measured. The content of total phenolic compounds was determined by Gutfinger (Gutfinger, 1981 ) Was modified and measured.

 That is, 1 ml of a 2% (w / v) Na2CO3 solution was added to 1 ml of the sample, and the mixture was reacted at room temperature for 3 minutes. Then, 0.2 ml of 50% Folin-Ciocalteu reagent was added and reacted at room temperature for 30 minutes. 1 ml of this reaction was taken and absorbance was measured at 750 nm. At this time, the standard concentration curve was prepared using EGCG (epigallocatechin gallate). Experimental results were expressed as means ± standard error (mean ± SEM) by repeating 3 times and compared by statistical treatment with Student's t-test. Statistical significance was determined when the p value was less than 0.05 (p <0.05).

As a result of this experiment, as shown in Fig. 2, the polyphenol content of the herbal extract of the conjugate obtained in Example 3 (5-stearate 3-valent sugar 2) was high.

< Experimental Example  3> MTT Assay (Cytotoxicity test)

Mouse macrophage Raw264.7 cells were seeded at 1 × 10 ⁴ cells / well in a 96-well plate and cultured in a CO2 incubator for 24 hours. After 24 hours, the culture medium was removed and the cells were treated with DMSO (final concentration 1%) together with the new culture medium and cultured for 24 hours. MTT solution (5 μg / mL) dissolved in PBS was added to a 96-well plate cultured for 24 hours and left in a CO2 incubator for 3 hours. When MTT forms blue formazan by mitochondrial dehydrogenase in the mitochondria of living cells for 3 hours, the supernatant is removed and 200 μL of DMSO is added to each well for shaking for 20 minutes. Absorbance was measured at 570 nm with an ELISA reader. Cell viability (%) was expressed using the following equation. Cell survival rate (%) = (absorbance of sample treated group / absorbance of control group) x 100

As shown in FIG. 3, other extracts except for gardenia (500 ppm and 1000 pp) showed no cytotoxicity.

< Experimental Example  4> Fibrinolytic activity (thrombolytic activity test)

Add 0.1 mL of thrombin (100 NIH unit / mL) dissolved in the same buffer to 5 mL of fibrinogen dissolved in 50 mM sodium phosphate buffer (pH 7.4) at a concentration of 0.5%, and add agarose agarose) solution was added and mixed. The mixture was poured into a petri dish (9 cm in diameter) and solidified at room temperature for 1 hour. A 3 mm diameter hole was drilled in the solidified fibrin-agarose gel, and the extracts obtained in Examples 2 and 3 were treated at each concentration. Plasmmin, a protein hydrolyzing enzyme, was used as a positive control and psychic rings were used as a negative control.

As shown in FIG. 4 and Table 1, the herbal extracts of the present invention obtained from Example 2 and Example 3, which contained fibrinolytic activity in a concentration dependent manner, Respectively.

Sample Treatment concentration Melting area Plasmin 1 U / mL, 10 [mu] L 13.85 cm2 6 per cent 4 100 ppm, 10 μL 2.01 cm2 6 per cent 4 400 ppm, 10 μL 6.15 cm2 5 levels per level 3 100 ppm, 10 μL 2.27 cm2 5 levels per level 3 400 ppm, 10 μL 8.04 cm 2 Psychic circle 100 ppm, 10 μL 1.54 cm2 Psychic circle 400 ppm, 10 μL 2.54 cm2

< Experimental Example  5> Of cell adhesion protein  Expression inhibition experiment

Experimental Example 5-1. Cell adhesion protein (ICAM-1) expression inhibition experiment

Cell adhesion protein (ICAM-1, Intercellular Adhesion Molecule-1) is known to be involved in vascular stenosis, thrombus formation, and blood circulation disorders. Immunocytochemistry was performed to measure the effect of inhibiting the expression of ICAM-1, a cell adhesion protein, in human umbilical vein endothelial cell (HUVEC) cells, a human umbilical vein endothelial cell line activated by TNF-α Respectively.

The HUVEC cells were seeded at 1 × 10 ⁴ cells / ml in 1 ml / well and placed in a 24-well plate at 12 ° C. for 24 hours. The samples (extracts obtained in Examples 2 and 3 ), Treated with 1 μg / ml of TNF-α and incubated at 37 ° C for 24 hours. The medium was washed with PBS, fixed with 4% formalin, and incubated with primary antibody ICAM-1 Cell Signaling Technology, MA, USA) and secondary antibody (Santa Cruz Biotechnology, CA, USA). Background staining with Mayer's hematoxylin.

As a result, as shown in FIG. 5, the normal human umbilical vein endothelial cell group (PBS treated group) showed low ICAM-1 expression but the TNF-α activated group (TNF-α treated group) (Reddish-brown labeled cells), and the expression of ICAM-1 in a concentration-dependent manner when treated with the sugar 6-valent 4 and 5-valent three-valent 2 obtained in Example 2 and Example 3 . In addition, ICAM-1 expression was not affected by treatment with the control group.

Experimental Example 5-2. Inhibition of Vascular Cell Adhesion Protein (VCAM-1) Expression

The vascular cell adhesion protein (VCAM-1, Vascullarr Adhesion Molecule-1) is known to be involved in vascular stenosis, thrombus formation, and blood circulation disorders. In order to measure the effect of suppressing the expression of vascular cell adhesion protein VCAM-1 in human umbilical vein endothelial cell (HUVEC) cells, a human umbilical vein endothelial cell line activated by TNF-α, the same method as Experimental Example 5-1 And immunocytochemistry was performed.

As a result, as shown in Fig. 6, the expression of VCAM-1 in the normal human umbilical vein endothelial cell group (PBS treated group) was low, but that of the TNF-a treated group (TNF-a treated group) (Reddish-brown labeled cells), and the expression of VCAM-1 was decreased depending on the treated concentration when the saccharide 6-valent 4 or 5-valent 3-valent 2 obtained in Example 2 or Example 3 was treated . In addition, VCAM-1 expression was not affected by treatment of the control group.

<Experimental Example 6> Inhibition of vaso-inflammatory enzyme expression

Experimental Example 6-1. Inhibition of vascular inflammation-inducing enzyme (COX-2) expression

Immunocytochemistry was performed to measure the inhibitory effect of COX-2 (Cyclooxygenase-2), which is a vascular inflammatory enzyme, in vivo in an inflammatory cell (Raw264.7 cell) model using LPS.

A 12-mm cover glass was placed on a 24-well plate, and RAW 264.7 cells were seeded at 1 × 10 ⁴ cells / ml in 1 ml / well, incubated at 37 ° C for 24 hours, The cells were incubated at 37 ° C for 24 hours. The cells were washed with PBS and fixed with 4% formalin. The primary antibody, COX-2 (Cell Signaling Technology, MA, USA) Antibodies were labeled with DAB (Santa Cruz Biotechnology, CA, USA) and developed with DAB. Background staining with Mayer's hematoxylin.

As a result, as shown in FIG. 7, the expression of COX-2 in the normal cell group (PBS treatment) was low, but about 50% or more cells (reddish brown labeled cells) were found in the inflammatory cell group (LPS treatment) It was confirmed that COX-2 expression was not inhibited at a concentration of 200 ppm but inhibited at a concentration of 1000 ppm in the cell group treated with the saccharide-free 6-level 4 or 5-level 3-level 2 prepared in Example 2 or 3. In addition, COX-2 expression was not affected by treatment of the control rings.

Experimental Example 6-2. Inhibition of vascular inflammation-inducing enzyme (5-LO)

In order to measure the inhibitory effect of 5-LO (5-Lipoxygenase) in vivo in the inflammatory cell (Raw264.7 cell) model using LPS, the immunoreactivity of 5-LO And immunocytochemistry was performed.

As a result, as shown in FIG. 8, the expression of 5-LO was low in the normal cell group (PBS treatment), but in the inflammatory cell group (LPS treatment) Cells) and the cells treated with 6-valent 4-glycoprotein 4 or 5-fold 3-valent 2 produced in Example 2 and 3 did not inhibit the expression of 5-LO at a concentration of 200 ppm, but were inhibited at a concentration of 1000 ppm . In addition, 5-LO expression was not affected by treatment of the control rings.

< Experimental Example  7> Vascular inflammation induction  enzyme( COX -2 and 5- LO ) On the expression of Western blotting )

Western blotting was performed to measure the inhibitory effect of the inflammatory enzymes COX-2 and 5-LO on in vivo expression in inflammatory cells (Raw264.7 cell) using LPS.

Inflammatory responses were measured by LPS-induced inflammatory cell model. Cells were lysed to determine total protein concentration, and 20 mg of total protein per well was electrophoresed SDS-PAGE) and then transferred to a nitrocellulose membrane. Then, the primary antibody and secondary antibody (COX-2, Cell Signaling Technology, MA, USA) or 5-LO (Santa Cruz Biotechnology, CA, USA) and enhanced chemiluminescence (ECL, Pierce, IL, USA) Respectively.

As a result, as shown in FIG. 9, in the Western blotting experiment, the expression of COX-2 and 5-LO proteins themselves was not affected regardless of the concentrations of 5-fold trinucleotide 2, .

< Experimental Example  8> Vascular inflammation induction  enzyme( COX -2)

In order to investigate the inhibitory effect of COX-2 (cyclooxygenase-2), a vascular inflammation inducing enzyme, arachidonic acid, which is a substrate of this enzyme, is used to produce PGF2, a precursor of prostaglandin, Were measured by ELISA.

10 μl of COX-2 enzyme, 10 μl of heme, and 950 μl of reaction buffer were added to a 1.5 ml tube, and 20 μl of the sample was added to the tube and stabilized at 37 ° C for 5 minutes. The reaction was stopped by adding 10 μl of arachidonic acid, reacting at 37 ° C for 2 minutes, and then adding 50 μl of 1 M HCl. Add 100 μl of SnCl2, mix well and leave at room temperature for 5 minutes. The reaction solution was diluted 2,000 times with EIA buffer 4,000 times and 50 μl was added to a 96-well plate coated with prostaglandin antibody. Then, 50 μl of PGs screening AChE tracer and 50 μl of antiserum were added and reacted at room temperature for 18 hours. The 96 wells were washed 5 times with buffer, 200 μl of Ellman's reagent was added, and the solution was developed for 60 minutes and then absorbance was measured at a wavelength of 410 nm. The inhibition of COX-2 activity was expressed by the value of IC50 (ppm), which is a 50% inhibitory concentration according to the manufacturer's method. Experimental results were expressed as means ± standard error (mean ± SEM) by repeating 3 times and compared by statistical treatment with Student's t-test. Statistical significance was determined when the p value was less than 0.05 (p <0.05).

The IC50 value, which is a concentration that inhibits 50% of COX-2, a vascular inflammation inducing enzyme, is shown in Fig. 6-valent 4-valent and 5-valent 3-valent valentine 2 were less effective than ibuprofen, a synthetic drug used as a representative COX-2 inhibitor, but significantly inhibited COX-2 activity compared to psychotropic drugs .

< Experimental Example  9> ACE  Enzyme inhibition

To examine whether the extracts obtained in Examples 2 and 3 inhibit the angiotensin-converting enzyme (ACE) enzyme responsible for the vasoconstriction action and induce blood pressure reduction and smooth circulation through blood vessel relaxation, saw. N-Hippuryl-His-Leu was used instead of angiotensin I, which is the substrate of ACE used in the experiment. ACE was extracted from the lung tissue powder of rabbits and used as an enzyme solution. The inhibition rate of hippuric acid produced by enzyme reaction was measured at 228 nm.

Samples were dissolved in distilled water to 3000 ppm and stored at 4 ° C until use. 21 (6) 811 (1986), and the present inventors conducted a modification of the method of Cho et al. (EK Cho, SK Yoo and YJ Choi, Inhibitory Effects of Maesaengi (Capsosiphon fulvescens) extracts on angiotensin converting enzyme and α-glucosidase. -818, 2011). The rabbit lung tissue powder (Sigma, USA) was adjusted to a concentration of 0.1 g / ml (w / v) of 0.1 M sodium borate buffer (pH 8.3) containing 0.3 M NaCl and then extracted at 4 ° C for 2 hours. Then, centrifugation was carried out at 4 ° C at 4000 rpm for 40 min, and the supernatant was used as the ACE enzyme solution. The substrate was prepared by dissolving hippuryl-histidyl-leucine (HHL) at a concentration of 5 mg / ml (w / v) in 0.1 M sodium borate buffer (pH 8.3) containing 0.3 M NaCl. For ACE inhibitory activity, 50 μl of ACE enzyme solution was added to 50 μl of sample, followed by preliminary reaction at 37 ° C for 5 minutes, followed by addition of 50 μl of substrate solution, followed by reaction at 37 ° C for 2 hours. 150 μl of 0.1 M HCl was added to stop the reaction, 750 μl of ethyl acetate was added, and the mixture was stirred for 15 seconds and centrifuged at 4 ° C and 3000 rpm for 5 minutes. The supernatant was completely dried and dissolved in 1 ml of distilled water and absorbance was measured at 228 nm. As a negative control, distilled water was used instead of crude enzyme solution, and enalapril maleate salt was used as a positive control. All control and experimental groups were repeated twice to calculate the ACE inhibition rate using the following formula. ACE inhibition rate (%) = [1 - (OD of ACE test / OD of ACE control) x 100

As a result, as shown in Table 2 and FIG. 11, it was found that the inhibition rate of the ACE enzyme was also increased as the final concentration of 6-valent 4-valent and 5-valent 3-valent 2 was increased.

Production Concentration (ppm) Final concentration (ppm) Inhibition rate IC ₅₀ 5 levels per level 3 3000 1000 86.7 ± 0.52 235.0 ppm 1500 500 66.2 ± 1.05 750 250 57.4 ± 2.79 375 125 37.2 ± 1.39 6 per cent 4 3000 1000 51.3 ± 2.61 896.3 ppm 1500 500 43.4 ± 2.09 750 250 34.1 ± 2.35 375 125 30.9 ± 2.09 Psychic circle 3000 1000 81.6 ± 0.26 395.0 ppm 1500 500 61.2 ± 0.35 750 250 48.3 ± 1.66 375 125 25.8 ± 0.17 Enalapril maleate salt
(Positive control group) 75 25 91.9 ± 0.78

< Experimental Example  10> Blood coagulation Related Parameters ( Thromboxane  B2) production inhibition

Experimental Example  10-1. Preparation of experimental animals and preparation of plasma

A total of 260-300 g of 7-week-old male Sprague Dawley rats (Sumataka, Korea) were purchased and adapted to the new breeding environment for 1 week. During the experiment, the animals were allowed to freeze the solid feed (Sumatoco, Korea) . The incubation room temperature was maintained at 20 ± 2 ° C and the relative humidity was maintained at 65 ± 5%, and light was adjusted to light for 12 hours. First, a normal diet was prepared as shown in Table 3, and a high-cholesterol diet was prepared as shown in Table 4.

Ingredient % (w / w) Casein (from milk) 20 Corn Starch 15 Sucrose 49 Cellulose 6 Corn Oil 5 Mineral Mixture 3.5 Vitamin Mixture One Choline Bitartrate 0.2 DL-Methionine 0.3 Cholesterol 0 Cholic acid 0 Total 100

Ingredient % (w / w) Casein (from milk) 20 Corn Starch 15 Sucrose 49 Cellulose 4.5 Corn Oil 5 Mineral Mixture 3.5 Vitamin Mixture One Choline Bitartrate 0.2 DL-Methionine 0.3 Cholesterol One Cholic acid 0.5 Total 100

First, the animal groups were divided into two groups, and the general diet and high cholesterol diet prepared in Table 3 and Table 4 were fed to these two groups of animals for two weeks. Then, each of the complex extracts prepared in Examples 2 and 3 was supplied with high cholesterol diet according to concentration as shown in Table 5.

Sample type Oral dose  / day (28 days ) A normal diet
( Normal diet ) sham DW High cholesterol diet
( High - cholesterol diet ) - ( Negative control group ) DW 6 per cent 4 -100 6 per cent 4 100 mg / kg body weight 6 per cent 4 -400 6 g per 4 400 mg / kg body weight 5 levels per level 3 -100 5 levels per 3 2 100 mg / kg body weight 5 levels per level 3 -400 500 mg / kg body weight Psychic circle -400 (positive control group) 400 mg / kg body weight

Twenty-four hours after the last feeding, the animals were anesthetized with ether. Blood was collected from the inferior vena cava and left at room temperature for 20 minutes. Centrifugation was then carried out using a high-speed centrifuge (Centricon T-42K, Italy) at 3,000 rpm for 15 minutes to obtain plasma .

Experimental Example  10-2. Experiment to inhibit the formation of blood coagulation factors

Thromboxane A2 (TXA2) is known to be involved in stroke, thrombosis, blood circulation disorder, angina pectoris and bronchial asthma due to platelet aggregation, vasoconstriction, and bronchoconstriction. TXA2 transformed with prostaglandin (PGG2 or PGH2) has a half-life of about 30 seconds and is chemically unstable. It is transformed into the final metabolite, Thromboxane B2 (TXB2), in the body. Thromboxane B2 contracts the blood vessels and collects platelets . &Lt; / RTI &gt;

To determine whether the extracts obtained in Examples 2 and 3 inhibit the production of blood coagulation-related factors (Thromboxane B2) using the plasma collected in Example 4, thromboxane B2 (TMA) was prepared by using TXB2 EIA kit (Enzo, USA) thromboxane B2, TXB2) were measured.

The standard TXB2 contained in the plasma or kit was diluted by the concentration in the test tube, and then 100 μl was added to a 96-well plate, and then the primary antibody (Goat anti-rabbit IgG antibody) And conjugate (alkaline phosphatase conjugated TXB2) was added thereto. To the reaction mixture was added a secondary antibody (rabbit polyclonal antibody to TXB2) and reacted at room temperature for 2 hours. Each well was washed three times with TBST, 200 μl of p-nitrophenyl phosphate solution was added, and reacted at room temperature for 45 minutes. After stopping the reaction, the absorbance was measured at a wavelength of 405 nm using an ELISA reader (Molecular Devices, USA). Experimental results were expressed as mean ± standard deviation (SEM), and were compared by Student's t-test. Statistical significance was determined when the p value was less than 0.05 (p <0.05).

As a result, as shown in Table 6 and FIG. 12, as the concentration of the herbal medicine extracts obtained in Example 2 and Example 3 (per 6-valent 4-valent and 5-valentinal 3-valent) was increased, blood coagulation-related factors (Thromboxane B2) was decreased.

Thromboxane B ₂ in plasma (pg / ml) Normal diet sham 451.6 + - 53.3 High-cholesterol diet (-) control 1033.4 ± 211.7 6 per cent 4-100 949.4 ± 160.1 Per 6th floor 4-400 881.4 ± 231.1 5 steps per 3-way 2-100 794.2 ± 198.3 5 steps per 3-way value 2-400 681.0 占 85.0 Mental Circle -400 824.1 + - 261.9

Claims (12)

35 to 65 wt% of Angelica gigas Nakai extract, 15 to 45 wt% of ethanol extract, and 5 to 35 wt% of ginger ethanol extract as an active ingredient.
delete delete delete The method according to claim 1,
Wherein said composition inhibits expression of a cell adhesion protein. The method according to claim 1,
Wherein said composition inhibits the expression of a vascular inflammation-inducing enzyme. delete delete delete delete delete delete

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