KR100988525B1 - A composition comprising the extract of complex herbsBDS as an active ingredient for preventing and treating atherosclerosis - Google Patents

Abstract

The present invention relates to a composition containing a complex herbal extract of Strychnos ignatii semen and licorice (Glycyrrhizae Radix) as an active ingredient. Specifically, the complex herbal extract of the present invention is TNF- in HASMC which is a human arterial smooth muscle cell. By confirming that effectively inhibit the cell migration induced by α, the composition can be usefully used as a pharmaceutical composition or health functional food for the prevention and treatment of atherosclerosis.

Strychnos ignatii semen, licorice (Glycyrrhizae Radix), complex herbal extract, arterial smooth muscle, arteriosclerosis

Description

A composition comprising the extract of complex herbs (BDS) as an active ingredient for preventing and treating atherosclerosis}

The present invention relates to a pharmaceutical composition or health functional food for preventing and treating atherosclerosis, which contains a complex herbal extract of Strychnos ignatii semen and licorice (Glycyrrhizae Radix) as an active ingredient.

Candrasekar B, et al., J. Biol Chem . 281 (22) pp 15099-15109, 2006

[2] Lee TH, et al., Mol . Cells , 23 (3) , pp 398-404, 2007

Kodali R, et al., Chemokines induce matrix metalloproteinase-2 through activation of epidermal growth factor receptor in arterial smooth muscle cells, Cardiovasc Res , 69 (3) pp 706-715, 2006

[4] Newby AC, et al., Molecular mechanisms in intimal hyperplasia, J. Pathol , 190 (3) pp 300-309, 2000

Cho A, et al., Matrix metalloproteinase-9 is necessary for the regulation of smooth muscle cell replication and migration after arterial injury, Circ . Res , 91 (9) pp 845-851, 2002

Lin SJ, et al., Salvianolic acid B attenuates MMP-2 and MMP-9 expression in vivo in apolipoprotein-E-deficient mouse aorta and in vitro in LPS-treated human aortic smooth muscle cells, J. Cell Biochem , 100 (2) pp 372-384, 2007

Cho A, et al., Mitogen-activated protein kinases mediate matrix metalloproteinase-9 expression in vascular smooth muscle cells, Arterioscler Thromb Vasc Biol , 20 (12) pp 2527-2532, 2000

Raffetto JD, et al., Matrix metalloproteinases and their inhibitors in vascular remodeling and vascular disease, Biochem Pharmacol , 75 (2) pp 346-359, 2008

[Document 9] Shin Gil-gu, Shin Chun Elementary School, Seoul, Hydrological History, p 705, 1982

Genevi Philippe, et al., Michel Fr About the toxicity of some Strychnos species and their alkaloids, Toxicon , 44 (1) pp 405-416, 2004

Document 11 The Committee of the Pharmacopoeia of the Ministry of Health of the people's Republic of China. Pharmacopoeia of the People's Republic of China, vol 1 , Chemical Industry Press , Beijing, p 38, 2000

[Ref. 12] Kim, Tae-Woon et al., The Effect of Combination Treatment of Ginseng and Ginseng on the Gastrointestinal Tract, Journal of Korean Oriental Internal Medicine , 15 (2) pp 27-36, 1994

[Reference 13] Cho, Yoo-Kyoung, 4, Influences on the Physiologic and Chemical Composition of the Korean Journal of Oriental Medicine, Journal of Korean Oriental Internal Medicine , 23 (1) pp 107-116, 2002

[Document 14] Jung-Seop Shin and Min-Kyo Shin, Doha Hyang-ja (Medicinal Medicine) , Yeonglimsa, pp 684-687, 1998

[15] Hyun-Jeong Yoon et al. 6, Induction of Apoptosis and Cell Cycle Arrest by SNU-1 Cells by Bodusan, Korean Journal of Herbology , 22 (2) pp 35-43, 2007

[16] Ahmed MD, et al., Restoration of transforming growth factor-beta signaling enhances radiosensitivity by altering the Bcl-2 / Bax ratio in the p 53 mutant pancreatic cancer cell line MIA PaCa-2, J. Biol. Chem , 277 (3) pp 2234-2246, 2002

Cheng EH, et al., BCL-2, BCL-X (L) sequester BH3 domain-only molecules preventing BAX- and BAK-mediated mitochondrial apoptosis, Mol Cell , 8 (3) pp 705-7511, 2001

Xu-Kun Deng, et al., The anti-tumor effects of alkaloids from the seeds of Strychnos nux-vomica on HepG2 cells and its possible mechanism, J Ethnopharmacology , 106 pp 179-186, 2006

19, Wu Yina, et al., The cytotoxicity induced by brucine from the seed of Strychnos nux-vomica proceeds via apoptosis and is mediated by cyclooxygenase 2 and caspase 3 in SMMC 7221 cells, Food and Chemical Toxicology , 45 pp 700-708, 2007

[20] Desai A, et al., Cytotoxicity and apoptosis enhancement in brain tumor cells upon coadministration of paclitaxel and ceramide in nanoemulsion formulations, J Pharm Sci , [Epub ahead of print], 2007

Jin UH, et al., Inhibitory effect of Salvia miltiorrhia BGE on matrix metalloproteinase-9 activity and migration of TNF-alpha-induced human aortic smooth muscle cells, Vascul Pharmacol , May 44 (5) pp 345-353, 2006

[22] Moon SK, et al., ERK1 / 2 mediates TNF-alpha-induced matrix metalloproteinase-9 expression in human vascular smooth muscle cells via the regulation of NF-kappaB and AP-1: Involvement of the ras dependent pathway, J. Cell Physiol , 198 (3) pp 417-427, 2004

23. Moon SK, et al., In vitro cellular aging is associated with enhanced proliferative capacity, G1 cell cycle modulation, and matrix metalloproteinase-9 regulation in mouse aortic smooth muscle cells, Arch Biochem Biophys , 418 (1) pp 39-48, 2003

Atherosclerosis causes vascular diseases such as stroke and coronary artery disease. In particular, angina and acute myocardial infarction caused by coronary artery disease have increased significantly over the last 20-30 years due to rapid social and economic development and westernized lifestyle. Arteriosclerosis is one of the most common circulatory diseases after hypertension, and it is one of the most important diseases in the national health as it accounts for a large portion of deaths along with cancer. Therefore, the importance of research on the pathogenesis and treatment of atherosclerosis is increasing.

Early lesions of atherosclerosis begin with the introduction of monocytes and T-cells into the blood vessel wall. Introduced monocytes differentiate into macrophage and further phagocytosis of lipids into foam cells, which accumulate and form a fatty streak in the vessel wall. Activated T-cells and foam cells express various inflammatory mediators, including cytokines and growth factors, sustain the inflammatory response and induce proliferation of smooth muscle cells, resulting in thickening of the vascular lining. Narrowing can cause heart disease such as angina. Macrophages and T-cells are involved in the formation of atherosclerotic plaques and are expressed by adhesion molecules, proinflammatory cytokines, chemokines, and metals. Matrix metalloproteinases (MMPs) increase the inflammatory response and migration, directly affecting the formation and rupture of atherosclerotic plaques (Chandrasekar B, et al., J Biol). Chem . 281 (22) pp 15099-15109, 2006; Lee TH et al., Mol . Cells . , 23 (3) , pp 398-404, 2007; Kodali R, et al., Chemokines induce matrix metalloproteinase-2 through activation of epidermal growth factor receptor in arterial smooth muscle cells, Cardiovasc Res , 69 (3) pp 706-715, 2006).

MMPs are tumor necrosis factor-α (TNF-α), interleukin-1α, and interleukin-1β, which are cytokines involved in atherosclerosis. ) And chemokine, IL-8, are mainly expressed in macrophages, vascular endothelial cells, and smooth muscle cells. The increased proteolytic activity of MMPs degrades the extracellular matrix (ECM) around smooth muscle cells, mediating the smooth muscle cell migration. Therefore, MMPs play an important role in proliferation of smooth muscle cells in the inner lining of blood vessels (intima), which causes many vascular diseases and deepens arteriosclerosis. In particular, MMP-2 and MMP-9 are reported to be involved in the smoothing and proliferation of smooth muscle cells. MMP-2 and MMP-9 are type IV collageanses (gelatinases) that degrade similar substrates. Pro-MMP-2 is always expressed in HASMCs and rarely expressed in MMP-2, pro-MMP-9, and MMP-9. Is increased by TNF-α or nerve growth factor (NGF) (Newby AC, et al., Molecular mechanisms in intimal hyperplasia, J. Pathol , 190 (3) pp 300-309, 2000; Cho A , et al., Matrix metalloproteinase-9 is necessary for the regulation of smooth muscle cell replication and migration after arterial injury, Circ Res , 91 (9) pp 845-851, 2002; Lin SJ, et al., Salvianolic acid B attenuates MMP-2 and MMP-9 expression in vivo in apolipoprotein-E-deficient mouse aorta and in vitro in LPS-treated human aortic smooth muscle cells, J. Cell Biochem , 100 (2) pp 372-384, 2007). In addition, the increase of MMP-9 by TNF-α is reported to be induced by the activation of mitogen-activated protein kinases (MAPKs) in cells (Cho A, et al., Mitogen-activated protein). kinases mediate matrix metalloproteinase-9 expression in vascular smooth muscle cells, Arterioscler Thromb Vasc Biol , 20 (12) pp 2527-2532, 2000).

In vivo, there is a tissue inhibitor metalloproteinase (TIMP), a protein that can regulate MMP. The imbalance between MMPs and TIMPs increases vascular smooth muscle cell flow activity and causes atherosclerosis and vascular disease (Raffetto JD, et al., Matrix metalloproteinases and their inhibitors in vascular remodeling and vascular disease, Biochem Pharmacol , 75 (2) pp 346-359, 2008). Therefore, attention is focused on finding an inhibitor of such MMPs.

Recently, interest in atherosclerosis prevention materials is increasing from natural resources, and if MMPs inhibitors are obtained from natural products, they can reduce side effects than artificial chemicals and are competitive in terms of price. In this study, we investigated molecularly and cellular biological methods of Bodu-san, which is expected to inhibit MMPs in natural products.

Strychnos ignatii semen refers to the seed of the evergreen shrub, an evergreen shrub belonging to the Loganiaceae. It is known to have analgesic, myrrhosis, hemostasis, hookworm, and detoxification in the private sector, and it has been used for abdominal pain, stomach ulcer and stomach cancer, and it takes about 0.3-0.9g at a time. According to the law, it is mixed with licorice as a powder, called Bodusan, and it has been used as an anticancer treatment or analgesic (Shin Gil-gu, Sinsibon Herbal Studies , Seoul, Hydrologic History, p 705, 1982). .

Bodu is also called guayan or citrus, and it is a seed of citrus and soaked in vinegar or rice water and chopped when it is flexible. Temper is very toxic and enters the heart, stomach and liver. It has the effect of analgesic, myrrhosis, hemostasis, hookworm, detoxification, so that abdominal pain, gastric ulcer, gastric cancer, alluvial abdominal pain, snakes, and goggles. Take it as a powder or pour it in oil and use it for the treatment. The dose is 0.3-0.9g per day. Bodu is similar to the properties of the warrior, the same medicinal herb, but contains more strychnine, brucine, volatiles, resins, pigments, nonvolatile oils, and bassorin. There is no milk or starch. Bodus are tonic-like or intriguing like a knight, and have been used as a substitute for knights because of their low price. Ancient records have been known to cure cholera. This is useful for certain types of heart disease and requires great care because of its high activity and strong toxicity. The detoxification to this is morphine (Genevi Philippe, et al., Michel Fr About the toxicity of some Strychnos species and their alkaloids, Toxicon , 44 (1) pp 405-416, 2004). In particular, STRIKININE and BRUSIN are strong toxic alkaloids that are present in plants of the genus Strychnos, which have been used early in Chinese medicine to treat neurological diseases, vomiting, joints and traumatic pain (The Committee of the Pharmacopoeia of the Ministry of Health of the people's Republic of China.Pharmacopoeia of the People's Republic of China, vol 1 , Chemical Industry Press , Beijing, p 38, 2000).

As a report through an experimental study, Kim reported that analgesic activity, prolonged sleep time, increased intestinal motility, suppressed gastric motility, suppressed ulceration, suppressed gastric injury, and suppressed gastric acid secretion in the combined in vivo experiment of opacic acid and bodu (Kim, Tae-Woon et al., Effect of Combined Administration of Glucose and Glucose on Gastrointestinal Tract, Journal of Korean Oriental Internal Medicine , 15 (2) pp 27-36, 1994). Joe had a decrease in strychnine according to Bodu's numerical method, and also a pepsin inhibitory effect along with inhibitory effects on pyloric ligation and indomethacin-induced ulcers and gastric secretion. (Cho Yu-kyung, 4 et al., Effects on the 毒性 and 消化器 system according to the 修治 Act of the Korean Journal of Oriental Medicine, Journal of Korean Oriental Internal Medicine , 23 (1) pp 107-116, 2002).

Licorice (Glycyrrhizae Radix) is a medicinal plant of the legume legume (Leguminosae), a perennial herb that is native to eastern Siberia (G. glabra var. Glandulifera ) and northeastern China. Flavonoids such as terpene-based saponins, glycyrrhizin, liquiritigenin and neooliquiritigenin are known. (Jeong-seop Shin and Min-Kyo Shin, Ambassador Dohae Hyangje (Medicinal Herbs ) , Younglimsa, pp 684-687, 1998).

Yoon confirmed that Bodusan induces apoptosis through the activation of caspases and mitochondrial pathway of human gastric cancer cell line SNU-1 (Yun et al. 6, Bodusan (寶). Induction of Apoptosis and Cell Cycle Arrest by SNU-1 Cells, Korean Journal of Herbology, 22 (2) pp 35-43, 2007; Ahmed MD, et al., Restoration of transforming growth factor-beta signaling enhances radiosensitivity by altering the Bcl-2 / Bax ratio in the p 53 mutant pancreatic cancer cell line MIA PaCa-2, J. Biol Chem , 277 (3) pp 2234-2246, 2002; Cheng EH, et al., BCL-2, BCL-X (L) sequester BH3 domain-only molecules preventing BAX- and BAK-mediated mitochondrial apoptosis, Mol Cell , 8 (3) pp 705-7511, 2001), has reported the anticancer activity of Bodusan by inhibiting proliferation of cells in the G1 phase of the cell cycle. Deng and Yin are the proliferation inhibitory mechanisms of Brucine HepG2 cells and SMMC 7221 cells, the main components of Bodu, via caspase-3 and COX-2-mediated cyclooxygenase-2 cells. Induced death and cell cycle arrest (Xu-Kun Deng, et al. The anti-tumor effects of alkaloids from the seeds of Strychnos nux-vomica on HepG2 cells and its possible mechanism, J. Ethnopharmacology , 106 pp 179-186. 2006; Wu Yina, et al., The cytotoxicity induced by brucine from the seed of Strychnos nux-vomica proceeds via apoptosis and is mediated by cyclooxygenase 2 and caspase 3 in SMMC 7221 cells, Food and Chemical Toxicology , 45 pp 700-708, 2007).

There have been several reports on Bodusan and Bodu, but their number is not high and the study on the mechanism of action is more insufficient. In particular, the study on anti-arteriosclerosis activity has not been disclosed or disclosed anywhere in the literature.

Therefore, the present inventors completed the present invention by confirming the anti-arteriosclerosis effect by measuring the percutaneous inhibitory activity of TNF-α-induced cells in HASMC, which is an arterial smooth muscle cell.

In order to achieve the above object, the present invention provides a pharmaceutical composition for the prevention and treatment of atherosclerosis containing a complex herbal extract of Strychnos ignatii semen and licorice (Glycyrrhizae Radix) as an active ingredient.

In addition, the present invention provides a health functional food for the prevention and improvement of atherosclerosis containing a complex herbal extract of Botry (Strychnos ignatii semen) and licorice (Glycyrrhizae Radix) as an active ingredient.

Complex herbal extracts as defined herein have a dry weight ratio (w / w) of beans and licorice of 1 to 10: 1 to 20, preferably 1 to 5: 1 to 10, more preferably 1: 3 to 5 It is characterized by

Extracts as defined herein include solvents selected from water, including purified water, lower alcohols having 1 to 4 carbon atoms or mixed solvents thereof, preferably 10-100% methanol, more preferably 60-90% methanol extract.

Atherosclerosis as defined herein is characterized by acting through an activity that inhibits the migration of cells induced by TNF-α in HASMC, which is an arterial smooth muscle cell.

Hereinafter, the present invention will be described in detail.

Complex herbal extract of the present invention can be prepared as follows. First step of mixing the dry weight ratio (w / w) of the bean and licorice in 1 to 10: 1 to 10, preferably 1 to 5: 1-5; A solvent selected from water containing about 1 to 5 times, preferably 3 times (w / v%) purified water, lower alcohols having 1 to 4 carbon atoms or mixed solvents thereof, preferably 10 to 100% of this weight A second step of extracting with methanol, more preferably 60 to 90% methanol at 15 ° C. to 100 ° C., preferably at 20 ° C. to 30 ° C. for 24 to 60 hours, preferably 30 to 50 hours; A third step of repeating the second step twice; Through the manufacturing method comprising the fourth step of filtration concentrated and freeze-dried can be obtained the complex herbal extract of the present invention.

The present invention provides a pharmaceutical composition for the prevention and treatment of atherosclerosis, which contains the complex herbal extract obtained by the above method as an active ingredient.

In addition, the present invention provides a health functional food for the prevention and improvement of atherosclerosis containing the complex herbal extract obtained by the above method as an active ingredient.

The pharmaceutical composition for the prevention and treatment of atherosclerosis containing the complex herbal extract of the present invention comprises 0.1 to 50% by weight of the complex herbal extract based on the total weight of the composition.

The pharmaceutical composition containing the complex herbal extract of the present invention may further comprise suitable carriers, excipients and diluents commonly used in the preparation of pharmaceutical compositions.

Pharmaceutical compositions containing the complex herbal extract of the present invention may be in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, oral formulations, external preparations, suppositories, and sterile injectable solutions, respectively, according to conventional methods. Can be formulated and used. Carriers, excipients and diluents that may be included in the composition containing the extract of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium Phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used. Solid form preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, which form at least one excipient such as starch, calcium carbonate, sucrose, etc. Or lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate 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 and liquid paraffin. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The amount of the combined herbal extract of the present invention may vary depending on the age, sex, and weight of the patient, but may be administered from 0.1 to 100 mg / kg once or several times daily. The dosage may also be increased or decreased depending on the route of administration, the severity of the disease, sex, weight, age, and the like. Therefore, the above dosage does not limit the scope of the present invention in any aspect.

The pharmaceutical composition may be administered to various mammals such as mice, mice, livestock, humans, and the like. All modes of administration can be expected, for example by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection.

The present invention provides a health functional food for the prevention and improvement of atherosclerosis comprising the complex herbal extract as an active ingredient. Foods to which it may be added include various foods, powders, granules, tablets, capsules, syrups, beverages, gums, teas, vitamin complexes, and health functional foods.

In addition, the extract may be added to food or beverage for the purpose of preventing the atherosclerosis. In this case, the amount of the composite herbal extract in the food or beverage may be added at 0.01 to 15% by weight of the total food weight, and the health functional beverage composition may be added at a ratio of 0.02 to 5 g, preferably 0.3 to 1 g, based on 100 ml. have.

The health functional beverage composition of the present invention is not particularly limited to other ingredients except for containing the extract as an essential ingredient in the indicated ratio, and may contain various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks. . Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And sugars such as conventional sugars such as polysaccharides such as dextrin, cyclodextrin and the like and xylitol, sorbitol, erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (tautin, stevia extract) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of such natural carbohydrates is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the composition of the present invention.

In addition to the above, the extract of the present invention is a variety of nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, such as flavoring agents, coloring and neutralizing agents (such as cheese, chocolate), pectic acid and salts thereof, alginic acid and its Salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks and the like. In addition, the extract of the present invention may contain a pulp for the production of natural fruit juices and vegetable drinks. These components can be used independently or in combination. The proportion of such additives is not so critical but is usually selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the extract of the present invention.

As described above, the complex herbal extracts of Strychnos ignatii semen and licorice (Glycyrrhizae Radix) of the present invention show an active effect of inhibiting the TNF-α-induced cell migration in HASMC, which is an arterial smooth muscle cell It can be used as a composition for the prevention and treatment of sclerosis.

Hereinafter, the present invention will be described in detail. However, the following Examples, Reference Examples and Experimental Examples are merely illustrative of the present invention, but the content of the present invention is not limited thereto.

Comparative example  One. Bodu  Preparation of Extract

Bodu (Strychnos ignatii semen) was selected from the Department of Control Science, Dongguk University, and added 3 times of 80% methanol to 100 g, extracted at 25 ℃ for 48 hours, and the process was filtered twice. Concentrated and lyophilized to give 62.4 g (yield 20.8%) of the sample of the present invention (hereinafter referred to as "SIS").

Comparative example  2. Preparation of Licorice Extract

Licorice (Glycyrrhizae Radix) was selected from the Department of Oriental Medicine, Dongguk University, and added 3 times 80% methanol to 400 g, extracted at 25 ℃ for 48 hours, this process was filtered twice Concentration and lyophilization gave 47.4 g (yield 15.8%) of the sample of the present invention (hereinafter referred to as "GR").

Example  1. Preparation of Complex Herbal Extracts

Strychnos ignatii semen and licorice (Glycyrrhizae Radix) were selected from the Department of Oriental Medicine, Dongguk University, 100g, 400g each, added 3 times 80% methanol, and extracted at 25 ℃ for 48 hours. This process was repeated twice, filtered, concentrated and freeze-dried to obtain 50.7 g (yield 16.9%) of the sample of the present invention (hereinafter referred to as "BDS") (see Fig. 1).

Reference Example  1. Preparation of experimental materials

1-1. Reagent Preparation

Purified muscle cell medium (Smooth Muscle Cell Medium, SMCM; (Sandiego, USA, catalog number: 1101) was purchased. Sodium Dodesyl Sulfate (SDS, 161-0302), Acrylamide ( 161-0100) and Bis (161-0201) were purchased from Bio-Rad (Hercules, USA), and the Aqueous One Solution Cell Priliferation Assay (MTS; G3580) kit and Griess Reagent System were manufactured by Promega (Madison, USA, G2930). All reagents used in the experiments were used above the analytical grade.

1-2. Cell culture

Human arterial smooth muscle cell lines (Human Aortic Smooth Muscle Cells; HASMCs) were distributed by ScienCell (Sandiego, Calif.) And cultured in SMCM. Each cell was incubated at 37 ° C., 5% CO ₂ conditions.

Experimental Example  One. MTS  Assay assay )

Desai A, et al., Cytotoxicity and apoptosis enhancement in brain tumor cells upon coadministration of paclitaxel and ceramide in nanoemulsion formulations , J. Pharm The experiment was performed as follows using the 5- (3-caroboxymeth-oxyphenyl) -2H-tetrazolium inner salt (MTS) analysis method described in Sci , [Epub ahead of print], 2007.).

This measures the conversion of MTS to formazan by mitochondrial dehydrogenases. 1 × 10 ⁴ / well HASMCs cells were dispensed into 96 month plates and wells were treated for 18 hours at different concentrations (0, 100, 300, 500, 700, 1000 μg / ml). 20 μl of MTS solution per well was added and reacted at 37 ° C., 5% CO ₂ incubator for 4 hours, followed by measurement of absorbance at 450 nm using a microplate reader (DYNEX, Opsys MR, USA). Cell viability relative to the negative control group (medicine 0 μg / ml) was not expressed as a percentage. In order to correct the absorbance of the drug by each concentration, the culture medium without the cells were cultured together to compare and correct the absorbance of the control group and the experimental group to express the cell survival rate as a percentage.

To evaluate the cytotoxicity of human smooth muscle cell HASMCs, Bodu acid, Bodu, and licorice were treated for 18 hours at different concentrations (0 to 700 μg / mL), followed by MTS assay. GR) had no cytotoxicity up to 700 μg / ml (Figs. 2A, 2C) and Bodu (SIS) had no decrease in cell viability up to 500 μg / ml and 85.7% at 700 μg / ml Survival of showed some cytotoxicity (FIG. 2B). Therefore, Bodusan, Bodu, and Licorice were all tested at 500 μg / mL, which does not affect cell viability.

Experimental Example  2. Yoo Ju  exam( Migration assay )

For measuring the inhibitory activity against the judo ability of HASMCs of each medicinal extract in Bodusan and Comparative Examples in Example 1 (Jin UH, et al., Inhibitory effect of Salvia miltiorrhia BGE on matrix metalloproteinase-9 activity and migration of TNF-alpha-induced human aortic smooth muscle cells, Vascul The assay described in Pharmacol , May 44 (5) pp 345-353, 2006) was performed using a transwell chamber as follows.

First, incubated with HNMCs at 500 ng / ml of TNF-α for 24 hours and then cultured to obtain a culture medium (TNF-α). It is a secretion secreted from cells by α stimulation, which includes various cytokines, chemokines, growth factors, etc.). After gelatin is coated on a polycarbonate filter of the upper chamber, cells are seeded at 1 × 10 ⁶ cells / ml in the upper chamber. 1 ml of conditioned medium is added to the lower chamber, followed by incubation for 24 hours in a 37 ° C., 5% CO ₂ incubator. After incubation, the filter is fixed with 4% paraformaldehyde solution, washed twice with PBS, and stained with hematoxylin & eosin (H &E; hematoxylin, MHS16; eosin, HT110216; SIGMA-ALDRICH, USA). After washing with PBS again, dehydrated in 95% ethanol, the filter was transferred to slide glass, sealed with a permount solution (SP15, SIGMA-ALDRICH, USA) and covered with a cover glass. The resident cells were observed and counted with a filter through an optical microscope (BX50, OLYMPUS).

     As a result, TNF-α treatment showed that the cell strain increased about 5.8 times, and the increased cell strain decreased by 68.0%, 21.6% and 52.6%, respectively. (FIG. 3). Bodusan and its constituents, Bodu and Licorice, all reduced the cellular suprapotency of HASMCs, and the most effective among them, Bodusan, was found.

Experimental Example  3. MMP Activity against -9 activity ) Measure

For measuring the activity of Bodusan and each of the medicinal extracts in Example 1 MMP-9 (Jin UH, et al., Inhibitory effect of Salvia miltiorrhia BGE on matrix metalloproteinase-9 activity and migration of TNF-alpha- induced human aortic smooth muscle cells, Vascul Pharmacol, May44 (5) pp 345-353 , 2006) gelatin jimo our assay (Gelatin zymography assay) was performed as described in the

In order to measure MMP-9 activity, cells were cultured by dispensing cells at 1 × 10 ⁵ / ml in a 24 well plate, and then replaced with a serum free medium for 16 hours. After pretreatment with each medicine for 1 hour, 100 ng / ml of TNF-α (TNF-α concentration was not 500 ng / ml) was treated for 24 hours. Each cell culture was collected and subjected to gelatin zymography. 20 μl of the culture is mixed with SDS-sample buffer without reducing agent, followed by electrophoresis, and the gel is soaked twice for 30 minutes in 2.5% Triton X-100. SDS was removed and washed twice with distilled water for 20 minutes. The washed gel was added to an incubation buffer (50 mM Tris-HCl; pH 7.6, 10 mM CaCl _{2 ,} 150 mM NaCl, 0.01% NaN ₃ ) and reacted for 24 hours in an incubator at 37 ° C., followed by 0.1% coomassie. The degree of gelatin degradation was observed by staining with brilliant blue solution (B0770, SIGMA-ALDRICH, USA) for 30 minutes.

      Density analysis of bands obtained through the Gelatin zymography assay was measured using GelDoc-It BioImaging System (UVP 97-0139-04, USA).

The results of the experiments were expressed as mean and standard deviation, and the significance test was performed by student's t- test using Sigma Plot (version 7.0 for Window).

In HASMCs, MMP-9 is known to increase its activity by TNF-α (Moon SK, et al., ERK1 / 2 mediates TNF-alpha-induced matrix metalloproteinase-9 expression in human vascular smooth muscle cells via the regulation of NF-kappaB and AP-1: Involvement of the ras dependent pathway, J. Cell Physiol , 198 (3) pp 417-427, 2004; Moon SK, et al., In vitro cellular aging is associated with enhanced proliferative capacity, G1 cell cycle modulation, and matrix metalloproteinase-9 regulation in mouse aortic smooth muscle cells, Arch Biochem Biophys , 418 (1) pp 39-48, 2003).

As a result, as shown in FIG. 4A, MMP-9 induced by TNF-α was significantly reduced by Bodu acid (BDS). In addition, as a result of comparing EGCG (positive control group; E4268, SIGMA, USA), which is a derivative of catechin, a component of green tea, which is well known as an MMP inhibitor, compared to the case where Bodusan was treated under the same conditions Was found to be nearly similar to the MMP-9 inhibitory activity of EGCG (FIG. 4B).

These results suggest that the induction of HASMCs by boduic acid may be due to the inhibition of MMP-9 activity. Boduic acid may have potential as an atherosclerosis treatment and prevention agent.

Although the formulation example of the composition comprising the complex herbal extract of the present invention will be described, the present invention is not intended to limit it, it is intended to explain in detail only.

Formulation example  One. Powder  pharmacy

BDS 20 mg

Lactose 100 mg

Talc 10 mg

The above ingredients are mixed and filled in an airtight cloth to prepare a powder.

Formulation example  2. Preparation of Tablets

BDS 10 mg

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

After mixing the above components, tablets are usually prepared by tableting according to the method for preparing tablets.

Formulation example  3. Manufacture of capsule

BDS 10 mg

3 mg of crystalline cellulose

Lactose 14.8 mg

Magnesium Stearate 0.2 mg

According to a conventional capsule preparation method, the above ingredients are mixed and filled into gelatin capsules to prepare capsules.

Formulation example  4. Preparation of injections

BDS 10 mg

Mannitol 180 mg

Sterile distilled water for injection 2974 mg

Na ₂ HPO ₄ 12H ₂ O 26 mg

It is usually prepared in the above ingredient content per ampoules (2 ml) according to the preparation method of injectables.

Formulation example  5. Liquid  Produce

BDS 20 mg

10 g of isomerized sugar

5 g of mannitol

Purified Water Proper Amount

Normally, each component is added to the purified water to dissolve according to the preparation method of the liquid solution, lemon flavor is added, the above ingredients are mixed, purified water is added, the whole is adjusted to 100 ml by the addition of purified water, and then filled into a brown bottle and sterilized. To prepare a liquid solution.

Formulation example  6. Preparation of dietary supplements

BDS 1000 mg

Vitamin mixture proper amount

70 μg of Vitamin A Acetate

Vitamin E 1.0 mg

Vitamin B1 0.13 mg

Vitamin B2 0.15 mg

Vitamin B6 0.5 mg

0.2 μg of vitamin B12

Vitamin C 10 mg

10 μg biotin

Nicotinamide 1.7 mg

50 μg folic acid

Calcium Pantothenate 0.5 mg

Mineral mixture

Ferrous Sulfate 1.75 mg

Zinc Oxide 0.82 mg

Magnesium carbonate 25.3 mg

15 mg potassium monophosphate

Dicalcium Phosphate 55 mg

Potassium Citrate 90 mg

Calcium Carbonate 100 mg

Magnesium chloride 24.8 mg

Although the composition ratio of the above-mentioned vitamin and mineral mixtures is a composition that is relatively suitable for the health functional food, the composition is mixed in a preferred embodiment, but the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional health functional food manufacturing method. Then, the granules may be prepared and used for preparing the nutraceutical composition according to a conventional method.

Formulation example  7. Manufacture of health drinks

BDS 1000 mg

Citric acid 1000 mg

100 g oligosaccharides

Plum concentrate 2 g

1 g of taurine

900 ml of purified water whole

After mixing the above components according to the conventional healthy beverage production method, and stirred and heated at 85 ℃ for about 1 hour, the resulting solution is filtered and obtained in a sterilized 2 L container, sealed sterilization and then refrigerated and stored Used to prepare the healthy beverage composition of the invention.

Although the composition ratio is a mixture of the components suitable for the preferred beverage as a preferred embodiment, the blending ratio may be arbitrarily varied according to the regional and national preferences such as the demand level, the demanding country, and the intended use.

[National R & D project supporting this invention]

[Task unique number]

       R13-2005-013-01000-0

[Name of Buddha]

Ministry of Science and Technology (Korea Science Foundation)

[Name of research project]

Cardiovascular Disease Natural Product Development Research Center

[Name of Research Project]

Elucidation of the mechanism of action of natural substances with cardiovascular disease prevention and treatment using in vivo model

[Host]

Korea Science Foundation, Dongguk University, Gyeongsangbuk-do

[Research period]

2005. 6. 1 ~ 2010. 2. 28

1 is a diagram illustrating a method of preparing a complex herbal extract (BDS), and (data were selected through three independent triplicate experiments).

      Figure 2 is a view showing the results of measuring the effect on the cell viability of HASMCs of complex herbal extract (BDS), licorice (GR), and bodu (SIS),

Figure 3 is a diagram showing the results of measuring the effect on the cytotoxicity of HASMCs after TNF-α treatment of complex herbal extracts (BDS), licorice (GR), and Bodu (SIS),

Figure 4 is a comparison of the effects on the MMP-9 enzyme activity of HASMCs after the treatment of complex herbal extracts (BDS), licorice (GR), and Bodu (SIS) compared with the known MMP-9 inhibitors It is a figure which shows the result of having measured the effect on the.

Claims (6)

A pharmaceutical composition for the prevention and treatment of atherosclerosis, which contains a complex herbal extract of Strychnos ignatii semen and licorice (Glycyrrhizae Radix) as an active ingredient. The pharmaceutical composition according to claim 1, wherein the complex herbal extract has a dry weight ratio (w / w) of 1 to 10: 1 to 20 of bovine and licorice. The pharmaceutical composition according to claim 1, wherein the extract comprises a solvent selected from water including purified water, a lower alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof. The pharmaceutical composition according to claim 1, wherein the extract comprises 0.1 to 50% by weight of the total weight of the composition. Health functional food for the prevention and improvement of atherosclerosis, which contains complex herbal extracts of Botry (Strychnos ignatii semen) and licorice (Glycyrrhizae Radix) as an active ingredient. 6. The dietary supplement of claim 5 which is a powder, granule, tablet, capsule, syrup or beverage.

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