KR20060099015A - Prevention and treatment of hypercholesterolemia containing Platicodin D - Google Patents

Abstract

The present invention relates to a prophylactic and therapeutic agent for hypercholesterolosis containing Platicodin D.

The composition of the present invention inhibits acyl coenzyme A: cholesterol acyltransferase (ACAT), which plays an important role in atheromatous atherosclerosis, the process of atherosclerosis, and the farnesoid X receptor (FXR). It acts antagonistically to reduce the risk of atheromatous atherosclerosis in the artery. Formation of cholesterol and insoluble polymers inhibits the absorption of cholesterol, resulting in hyperlipidemia, hypercholesterolemia, coronary arteriosclerosis, It can be useful for the prevention and treatment of cholesterol-related diseases such as hypertension, myocardial infarction, angina pectoris, stroke, cerebral hemorrhage, peripheral vascular disease.

Description

Composition containing Platicodin D for the prevention and treatment of hypercholesterolemia}

1 is a diagram showing changes in body weight (platicodine D dosages 0 (▽), 15 (●), 30 (○) and 50 (▼) mg / kg, HC-free diet in HC diet group) Group (■)).

Figure 2 is a diagram showing the effect of inhibiting ACAT activity of Platicodin D (ACAT-1 (●), ACAT-2 (○)).

3 is a diagram showing the antagonizing effect on the FXR activity of Platicodin D (Platincodine D untreated (●), 40 nM Platicodin D treatment (○)).

4 shows polymer formation between cholesterol and Platicodin D. FIG.

The present invention relates to a prophylactic and therapeutic agent for hypercholesterolosis containing platycodin D.

95% of the fat in human food is trigriceride, and the rest is cholesterol and phospholipids. Cholesterol is a major component of cell membranes and lipoproteins, and is a precursor of bile acids, steroid hormones, and vitamin D _{3. It} is synthesized in most tissues except brain tissue and occurs most actively in the liver. In adults, the amount of cholesterol biosynthesized in the body per day is about 9-11 mg / kg body weight, which is much higher than the amount supplied by meals. Depends on.

Diseases related to cholesterol metabolism are mainly caused by the accumulation of cholesterol in the blood and tissues. Fats consumed through food are absorbed mainly in the duodenum and jejunum, and these fats are gradually absorbed by the emulsification of bile salts secreted from the liver before absorption. It is changed to a small fat spot. They then break down the action of lipase secreted by the pancreas into free fatty acids, monoacylglycerols or diacylglycerols, free cholesterol, and lysophosphatidyl choline. Among them, water-soluble glycerol and lysophosphatidyl choline are easily absorbed, but the remainder is dissolved in hydrophobic and hydrophilic mixed micelles before being absorbed. These colloids consist mainly of monoacylglycerols, fatty acids, bile salts and small amounts of cholesterol, and the role of bile acids is essential for their formation. Lipids absorbed in the form of colloidal particles are esterified in enterocytes and converted into triglyceride and esterifide cholesterol forms and then combined with apolipoprotein to form chylomicrons. do.

In this process, cholesterol that is not dissolved into the colloid is excreted through the intestine. On the other hand, endogenous and exogenous cholesterol present in the liver is partially secreted into the blood lipoprotein (lipoprotein) constituents and the remainder is converted into bile acids (cholic acid and chenodeoxycholic acid) is secreted into the small intestine with a small amount of phospholipids and cholesterol. Bile acids are used in the small intestine to form lipid colloids, and over 95% of them are recycled to the liver and the rest are excreted with feces. In this case, farnesoid X-receptor (FXR), a nuclear hormone receptor present in the liver, also called bile acid receptor, is known to act. FXR is activated by bile acids to produce bile acids, a form of cholesterol excretion, through feedback control to eliminate excess cholesterol and make cholesterol better melted to promote absorption.

Excess plasma cholesterol that has not been used or excreted in the body is known as a risk factor for diseases of the cardiovascular system, such as hypercholesterolemia and atherosclerosis (Levy, 181; Gurr, 1988; Gordon et al. , 1981). The amount of cholesterol that is reabsorbed in the intestine or synthesized in tissues can be regulated by negative feedback involving HMG-CoA (3-Hydroxy-3-methylglutaryl- Coenzyme A) reductase. (Gordon et al ., 1987). That is, HMG-CoA reductase activity and LDL (low density lipoprotein) -receptor synthesis are inhibited when the amount of cholesterol flowing into the cell is high, and HMG-CoA reductase activity is promoted when the amount of cholesterol flowing into the cell is low. Increasing LDL-receptor synthesis allows proper maintenance of intracellular cholesterol homeostasis. Blood cholesterol moves in the form of a macromolecule called lipoprotein, of which about three quarters are contained in LDL and blood LDL cholesterol concentrations are known to be proportional to the risk of atherosclerosis (Golden et al. , 1981; Frostegard et al . , 1990). Excess cholesterol is ultimately left to meet the needs of the cell, which is left in the blood, mainly contained in LDL particles, and wanders to the endothelial membrane with monosite and microphages to convert into foam cells. The foam bacteria eventually lead to a heart attack by blocking the arteries (Frostegard et al ., 1990).

In contrast, the high density lipoprotein (HDL) concentration in blood involved in cholesterol transport in cholesterol metabolism is inversely related to the incidence of atherosclerosis (Brown et al., 1986). Hypercholesterolemia is well known as a risk factor for coronary artery disease (Wald & Law, 1994, 1995). The purpose of the prevention and treatment of hypercholesterolemia is to remove excess cholesterol through normal lipid metabolism in the body. According to research reports of developed countries, cerebrovascular and cardiac diseases, which account for more than 30% of all mortality rates, are vascular diseases represented by cerebral hemorrhage, cerebral thrombosis, heart failure, and myocardial infarction. It is actively underway.

Two important enzymes in the regulation of cholesterol metabolism, which are present in liver tissue and affect cholesterol synthesis and storage, are HMG-CoA reductase (3-hydroxy-3-methylglutaryl coenzyme A, EC 1.1.1.34) and ACAT (acyl coenzyme A: cholesterol acyltransferase, EC 2.3.1.26). HMG-CoA reductase is an important rate limiting enzyme that regulates cholesterol synthesis in the liver. ACAT is an enzyme that esterifies the free cholesterol of the small intestine, liver and other tissues. Intestinal absorption of dietary cholesterol and VLDL (very) in the liver low density lipoprotein) -cholesterol secretion and arteriovascular cholesterol accumulation (Helgerud, 1981; sucking & stange, 1985). To date, reports of inhibitors of these two enzymes have shown antihypercholesterolemic effects as well as cholesterol lowering effects in various experimental animals including humans (Alberts et al ., 1980; Amin et al ., 1993; Alberts , 1988; Fujioka et al. , 1995; Schnizer-polokoff et al ., 1991; Largis et al ., 1989; Heidner et al. , 1983; William et al ., 1989; Roth et al. , 1992; Sugiyami et al ., 1995).

In this regard, many new drugs that are expected to be effective are being synthesized or developed. Looking at the antihyperlipidemic drugs developed and marketed up to now, first, LDL-cholesterol has been shown to have an effect of inhibiting the absorption of cholesterol and promoting its catabolism and excretion. There are cholesterol derivatives such as Cholestyramine, Cholestipol, Clofibrate, and Gemfibrozil (Stein, 1994) that can be lowered at. In addition, lovastatin (Havel, Havel, 1987), simvastatin (Illingworth et al, 1987), pravastatin (Kavaami et al . 1986), inhibitors of HMG-CoA reductase involved in cholesterol biosynthesis, have been developed. It is sold to. Recently, the development of new drugs through the study of substances with antagonism targeting FXR has been an issue.

However, these drugs, which have been developed so far, have the advantage of lowering cholesterol levels but have serious side effects and are not suitable for ideal drug therapy (Illingworth, 1988). While attempting to reduce the incidence of heart disease such as atherosclerosis by reducing biosynthesis (Grundy et al., 1981) , this side effect is severe and is prohibited in some countries.

Platycodin D is a type of saponin and is isolated from saponins that are found on the street. Gilkyung is mainly used as a herbal medicine as the root of bellflower, and is used as a medicine for cough, gallstones, expectorant, nasal congestion, cold, tonsillitis and pneumonia.

In general, saponins are classified into triterpenes and steroids as aglycones and a group of compounds having glycoside bonds with sugars. In addition, these saponin groups can be distinguished according to saponinin, of which the sagittal saponins belong to the pentacyclic oleanane-type triterpene saponin. Specifically, about 10% of triterpene saponins of about 10% are contained in the pathogenic saponin. Among these saponins, glycosides of platyodigenin are Platicodin A, C, D, D ₂ and two species. Monoacetate, Platicodin D ₃ has been reported (Rossi M et al. , Chem. Pharm. Bull 16 (11): 2300, 1968). On the other hand, as an immunopharmacological study of Gil-Kyung, Michinori et al. Reported that 70% methanol extract and its fractions were effective in phagocytic activity through carbon clearance in mouse reticuloendothelial tissues ( Michinori et al ., Shoyakugaku zasshi 40 (4): 367, 1986). The results suggest that crude placodiulin and crude placodi saponins promote phagocytosis of reticulose tissue. Namba et al . Also reported on the anticancer activity of inulin extracted from Gilkyung , which increased the anticancer activity when inulin was treated with mitomycin C (Namba et al ., Shoyakugaku zasshi). 40 (4): 375, 1986).

As a result of investigating the physiological activity of the saponin, it is reported that each activity is different according to the structure of saponin. Especially, among these saponins, substances called platicodine D and platicodine D ₃ induce inflammation, COX (cyclooxygenase)- 2 has been reported to indirectly inhibit (Kim YP et al. , Planta Med 67: 362-364, 2001) and has also been suggested as a treatment for various airway-related diseases by increasing mucin in the airways. Shin CY et al., Planta Med 68 (3): 221-225, 2002. Saponins with physiological activity against chronic anti-inflammatory effects are also known to have anticancer effects on certain cancer cells.

The inventors of the present invention, while studying various physiological activities of Platicodin D, Platicodin D inhibits acyl coenzyme A: cholesterol acyltransferase (ACAT) and antagonistically to the Farnesoid X receptor (FXR). The present invention completed the present invention by forming a cholesterol and an insoluble polymer, reducing the cholesterol content in the blood, liver and excreta, and can be useful as a preventive and therapeutic agent for diseases related to hypercholesterolemia.

The present invention is to provide an agent for the prevention and treatment of hypercholesterolosis containing Platicodin D as an active ingredient.

In order to achieve the above object, the present invention provides a high cholesterol prevention and treatment agent containing Platicodin D represented by the following formula (1) as an active ingredient.

Platicodin D used in the present invention is separated and purified from Platicodi Radix using a known method (Korean Patent Registration No. 10-0295366).

Platicodine D according to the present invention reduces weight and food intake when administered to mammals with hypercholesterolemia and lowers cholesterol content in blood, liver and feces. It also inhibits acyl coenzyme A: cholesterol acyltransferase (ACAT), which plays an important role in atherosclerotic aatherogenesis in the atherosclerotic process, and is antagonistic to the farnesoid X receptor (FXR). It acts to reduce the risk of atheromatous atherosclerosis. Platicodine D is expected to form cholesterol and insoluble polymers to inhibit the absorption of cholesterol.

Therefore, the composition containing Platicodin D as an active ingredient is useful for the prevention and treatment of cholesterol-related diseases such as hyperlipidemia, arteriosclerosis, hypertension, myocardial infarction, angina pectoris, stroke, cerebral hemorrhage, peripheral vascular disease as well as obesity Can be.

The composition of the present invention may further contain at least one active ingredient exhibiting the same or similar function in addition to the Platicodin D.

The composition of the present invention may be a pharmaceutical composition and may be used in the form of a general pharmaceutical formulation.

That is, Platicodin D of the present invention may be administered in various oral and parenteral dosage forms during actual clinical administration, and when formulated, the usual fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc. Prepared using diluents or excipients. 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 (Sucrose) or lactose (Lactose), gelatin, etc. are mixed and prepared. In addition to simple excipients, lubricants such as magnesium styrate 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 the suspension solvent, 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. In addition, calcium or vitamin D ₃ may be added to enhance the efficacy as a cholesterol lowering agent.

The dosage of the composition of the present invention varies depending on the body weight, age, sex, health condition, diet, time of administration, method of administration, excretion rate and severity of the disease, the daily dosage of Platicodin D 0.1 to 100 mg / kg, based on the amount, may be administered 1 to 6 times per day.

The composition of the present invention can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy and biological response modifiers to lower cholesterol.

The composition of the present invention may be in the form of a health food for the purpose of preventing and treating hypercholesterolemia. When the platicodine D of the present invention is used as a food additive, the platicodine D may be added as it is or used with other food or food ingredients, and may be appropriately used according to a conventional method. The blending amount of the active ingredient can be suitably determined according to the purpose of its use (prevention, health or therapeutic treatment). However, in the case of long-term intake for health and hygiene or health control purposes, the amount may be below the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount above the above range. It is certain.

There is no particular limitation on the kind of food.

The health beverage composition of the present invention may contain various flavors or natural carbohydrates, etc. as additional components, as in the general beverage. The above-mentioned natural carbohydrates are glucose, monosaccharides such as fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin, sugar alcohols such as xylitol, sorbitol and erythritol. As the sweetening agent, natural sweetening agents such as tautin and stevia extract, synthetic sweetening agents such as saccharin and aspartame, and the like can be used. The proportion of the natural carbohydrate is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 ml of the composition of the present invention.

In addition to the above, the composition of the present invention includes various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, And a carbonation agent used for the carbonated beverage.

Hereinafter, preferred examples and experimental examples are presented to help understand the present invention. However, the following Examples and Experimental Examples are provided only to more easily understand the present invention, and the contents of the present invention are not limited by the Examples.

Values were expressed as mean ± standard deviation (SD), and mean differences between groups were analyzed using one-way ANOVA and independent t -tests.

Example 1 Preparation of Platicodin D

100 kg of Platicodi Radix samples were extracted with methanol and partitioned sequentially with n-hexane, chloroform, ethyl acetate and n-butanol. The n-butanol fraction after fractionation was subjected to Diaion HP-20 resin (Mitsubishi Chemical Corporation, Japan) column to separate Platicodin D, eluted with 60-80% methanol and 90 g / ml of crude saponin Got. Crude saponin was further purified using silica gel (Merck, Germany) chromatography to obtain purified Platicodin D. The procedure was repeated several times until the appropriate purity of Platicodin D was obtained. Purified Platicodin D was identified based on the R _f , FAB-MS (= 1225.38) and ¹³ C-NMR spectral values of known Platicodin D samples. Purity was measured using an HPLC chromatogram (Agilent, Palo Alto, CA) of ZORBAX SB-Aq ODS C ₁₈ equipped with an ELSD detector (Sedex 75, Sedere, France).

Poly obtained divided new D (D polygalacin), Plastic Tycho Dean A (platycodin A) and plastisol Tycho Dean D ₃ as other saponins by-product in this separation step, such as Plastic Tycho Dean D (platycodin D _3).

Experimental Example 1 Experiment for Measuring Weight Change by Platicodin D

In order to confirm the cholesterol-lowering effect of Platicodin D, high cholesterol mice were made, and their weight change and feed intake were measured.

Oral Administration of Platicodin D to High Cholesterol ICP Mice

High cholesterol (HC) feed was prepared by including 1% cholesterol and 0.2% cholic acid (Sigma-Aldrich, St, Louis, MO) in the granular general feed. HC-free feed consisted of granular general feed. Both feeds were prepared by the same preparation method.

Male ICR mice (6 weeks old) were purchased from Seoul National University Animal Center. Animals were bred at constant temperature (21-23 ° C.) and state humidity (50-60%) in a 12 to 12 hour contrast cycle. The animals were given regular feed and water for one week before the experiment.

After feeding the animals with HC feed for a week, only animals with blood cholesterol above 200 mg / dl to be used in the experiment were selected. 24 mice selected were randomly divided into four groups (n = 6) to make three Platicodin D experimental groups and one HC control group. For the three experimental groups, 15, 30 or 50 mg of Platicodin D per kg body weight of the mice was administered orally once a day for 8 weeks. For the HC control group, 0.9% physiological saline was administered instead of Platicodin D as a negative control group. The normal control group fed the HC-free diet (n = 6) was used for comparison and fed the same amount of 0.9% saline instead of Platicodin D.

All procedures of animal testing were performed at Seoul National University in accordance with the Policy and Regulation for the Care and Use of Laboratory Animals.

Determination of weight and feed intake

Weight and feed intake were recorded twice a week. Feed intake efficiency of each group was recorded twice a week by monitoring the feed consumption (g) in each kennel for two consecutive days, and was calculated as the number per mouse per day.

result

Oral administration of Platicodin D to hypercholesterolemic ICR mice for 8 weeks resulted in a significant decrease in body weight (FIG. 1). In the three experimental animals treated with Platicodin D at 15, 30 or 50 mg / kg, 11.2 ± 5% ( p <0.01), 11.7 ± 5% ( p <0.01) and 23.4 ± 7.9%, respectively, compared to the HC control group. The weight of ( p <0.0001) was decreased. The group fed HC diet alone showed no change in body weight compared to the HC-free group, indicating that cholesterol itself had no significant effect on weight gain.

Similar reductions were also seen in the Platicodin D treated group in feed intake (Table 1). Contrary to previous reports, the researchers demonstrated a significant reduction in feed consumption, concentration-dependently. This trend was most noticeable during the first two weeks of the experiment. At the end of the experimental period, the feed consumption gradually decreased to the value of the control group except for the 50 mg / kg test group, and the feed intake remained somewhat lower than that of the control group throughout the experimental period.

Changes in Feed Intake During Platicodine D (PD) Administration (n = 6) Time (week) % Against control One 2 3 7 8 Control (HC) 100 ± 4.2 100 ± 5.9 100 ± 4.5 100 ± 1 100 ± 16.0 30 mg / kg PD (HC) 54.4 ± 0.2 ** 74.0 ± 12.2 * 68.2 ± 1.4 ** 80.4 ± 3.0 * 80.0 ± 4.0 50 mg / kg PD (HC) 42.0 ± 1.2 *** 65.4 ± 5.8 ** 53.1 ± 3.2 ** 65.0 ± 3.0 ** 67.9 ± 2.0 * Normal group (HC-free) 79.6 ± 12.7 75.1 ± 2.5 * 72.2 ± 0.2 ** 83.2 ± 16.8 82.5 ± 15.0 The figures are expressed as mean ± standard deviation (SD) and are significant at * p <0.05 and ** p <0.01 by independent sample t-test.

Experimental Example 2 Determination of Cholesterol Levels in Serum, Liver, and Excretion of Platicodin D

Collection of serum, liver and feces

At the end of the experiment animals were fed for at least 2 hours after Platicodin D administration and then stopped for 14 hours. Blood samples were collected by collecting blood around the eyes, stored at room temperature for 2 hours, and then centrifuged at 3,500 × g for 10 minutes to obtain serum. Serum was stored at −81 ° C. until analysis. After the blood collection, the animals were euthanized by cervical separation. The liver was cut off and weighed and stored at -81 ° C for the experiment. Excretion was collected 48 hours before the end of the experiment. Collected feces were lyophilized and weighed.

Measurement of serum parameters

The activity of serum alanine amino-transferase (ALT) and aspartate amino-transferase (AST), serum triglyceride (TG) and total cholesterol (TC) concentrations It was measured using a kit (Eiken Chemical Co. Ltd., Tokyo, Japan) available. Both analyzes are colorimetric methods that detect high-saturation end products at wavelengths of 490-520 nm using microplate absorbers (Molecular Devices, Sunnylvale, Calif.).

The concentration of HDL-cholesterol (HDL-C) was measured using a Hitachi-747 auto-analyzer (Hitachi-747 auto-analyzer, Hitachi, Co., Tokyo, Japan) for HDL-C diagnostic reagents (Kyowamedex Co.Ltd, Japan). Measured by. The concentration of LDL- cholesterol (LDL-C) is -7170 Hitachi Auto-Analyzer using (Hitachi-7170 auto-analyzer, Hitachi, Co., Tokyo, Japan) , Roche 2 ^nd generation LDL-C Plus (Roche 2 ^nd generation LDL-C plus) reagent.

Lipid Extraction and Separation

Lipids were extracted from liver and feces. Samples were extracted with an ultrasonicator for 5 minutes using chloroform and methanol (2: 1, 1 ml per 1 mg of sample). Distilled water was added to the supernatant after centrifugation at 3000 x g for 10 minutes to remove insoluble matter, and the lower layer was collected and dried in the presence of nitrogen. The dried extract was dissolved in 100 μl of chloroform per 10 mg and separated by a solid phase extraction (SPE) column (Alltech, Deerfield, IL) combined with aminopropyl. The aminopropyl column (500 mg) was first washed twice with 4 ml n-hexane in vacuo (˜10 kPa) using a vacuum manifold (Alltech, Deerfield, IL). A lipid extract dissolved in 100 μl of chloroform was injected into the column in vacuo, and the column was eluted with 6 mL of chloroform: 2-propanol (15: 1, v / v) to separate neutral lipids and contained neutral lipids. The eluent was dried in the presence of nitrogen.

Measurement of Neutral Lipids

For accurate quantitative analysis, lipids are liChrosphere 100 Diol column (5 μm, 4 × 250 mm, Merck), Hitachi pump (L-6200), evaporated light-scattering detector, Sedere, Sedex 75, Vitry-sur-Seine, France) and HPLC equipped with auto injector (Shimadzu, SIL-9A). Elution solvent A: hexane-acetic acid (99: 1, v / v) and elution solvent B: hexane-isopropanol-acetic acid (84: 15: 1, v / v / v) were used as a suitable elution system. Separation conditions were 0-25 minutes (12-100% B), 25-26 minutes (100% B), 26-29 minutes (100-12% B), after which the flow rate was 1 ml / min, column temperature 50 ℃, 12% 10 minutes with N ₂ partial pressure in the temperature detector 50 ℃ and 2 bar (bar) was homogenized as elution solvent B.

result

(1) Biochemical Analysis of Serum

As shown in Table 2, the levels of TC, LDL and TG of the serum HC diet group were significantly higher than those of the HC-free group, while HLD decreased. This result indicates that hypercholesterolemia caused by feeding cholesterol in the diet was effectively measured by ICR mice. Platicodine D administration at 15, 30 and 50 mg / kg reduced the elevated cholesterol levels to 65.5%, 73.7% and 79.6% of the HC control, respectively. Moreover, the concentration of low density lipoprotein cholesterol (LDL-C) was reduced to 57.8%, 56.8% and 43.9% of the HC control, respectively. However, there was no noticeable change in the concentration of high density lipoprotein cholesterol (HLD-C) in most of the Platicodin D-treated animals. Table 2 also shows that Platicodin D administration reduced elevated TG concentrations to 65.4-81.0% of HC controls.

Serum parameters measured 8 weeks after Platicodin D (PD) administration (n = 6) Parameter 15 mg / kg PD 30mg / kg PD 50 mg / kg PD Control Normal Diet HC HC HC HC HC-free TG (mg / 100ml) 96.1 ± 29.4 ** 94.5 ± 41.0 * 116.8 ± 20.7 * 144.6 ± 32.5 110.0 ± 20.8 * TC (mg / 100ml) 95.0 ± 13.0 ** 112.9 ± 8.1 ** 121.1 ± 24 152.8 ± 35.2 116.3 ± 12.5 * HDL-C (mg / 100ml) 48.4 ± 7.9 66.0 ± 22.8 61.8 ± 24.8 62.2 ± 6.1 74.9 ± 8.6 LDL-C (mg / 100ml) 40.8 ± 20.0 * 40.1 ± 28.5 * 31.0 ± 9.6 * 70.6 ± 28.3 60.8 ± 44.8 ^a HDL-C / LDL-C 1.6 ± 0.6 3.9 ± 2.7 2.1 ± 0.2 ** 1.0 ± 0.4 2.2 ± 1.0 *** ^a TC / HDL-C 2.1 ± 0.4 1.9 ± 0.7 1.9 ± 0.3 2.6 ± 0.5 1.5 ± 0.2 *** The figures are expressed as mean ± standard deviation (SD) and are significant at * p <0.05 and ** p <0.01 by independent sample t-test. ^a : The average of each value was used.

(2) biochemical analysis of liver

Platicodine D administration significantly reduced liver TG and cholesterol levels (Table 3). TG decreased to 43-68% in the groups of different PD doses. Changes in cholesterol content are represented by cholesterol esters (CE), which decreased to 48-60% in all experimental groups. In contrast, free cholesterol (FC) content remained largely unchanged in either experiment or HC control. Taken together, the CE / FC level in the HC control group improved to 4.06 (0.97 ± 0.11 in the HC-free group), while the Platicodin D treated group with elevated CE / FC ratio decreased significantly to 2.06-2.31. On the other hand, abnormalities were observed in serum ALT and AST activity. In the case of AST, there was a slight increase in the HC control group relative to the HC-free group and a marked decrease in AST activity with Platicodin D treatment, resulting in 26-27 in 15 and 30 mg / kg Platicodine D administered animals. A decrease of% was shown. In addition, liver mass was also slightly reduced in Platicodin D experimental animals, and the HC control liver mass was 1.98 ± 0.18 g, compared to 1.47 ± 0.38 g of the 15 mg / kg Platicodin D treated group.

Liver levels (n = 6) 8 weeks after Platicodine D (PD) administration Parameter 15 mg / kg PD 30mg / kg PD 50 mg / kg PD Control Normal Diet Method HC HC HC HC HC-free Liver mass (g) 1.47 ± 0.38 * 1.83 ± 0.14 1.81 ± 0.23 1.98 ± 0.19 1.84 ± 0.16 ^# TG (㎎ / g liver weight) 12.19 ± 2.19 15.84 ± 0.30 6.01 ± 3.02 * 19.67 ± 9.14 15.80 ± 4.41 ^# CE (㎎ / g liver weight) 14.36 ± 2.01 11.08 ± 5.89 * 14.19 ± 3.27 * 27.71 ± 7.84 6.80 ± 2.62 ^# FC (㎎ / g liver weight) 6.26 ± 0.87 5.36 ± 0.99 6.74 ± 2.41 6.74 ± 0.70 6.85 ± 2.05 ^& CE / FC 2.31 ± 0.29 * 2.09 ± 1.05 * 2.28 ± 0.84 ** 4.06 ± 0.76 0.97 ± 0.11 ^$ ALT (KU / l) (serum) 19.8 ± 0.5 * 19.1 ± 7.1 22.0 ± 1.2 * 25.1 ± 1.5 20.4 ± 2.7 * ^$ AST (KU / l) (serum) 42.6 ± 1.5 *** 42.3 ± 3.4 ** 57.6 ± 3.2 * 79.2 ± 3.2 57.0 ± 2.7 The figures are expressed as mean ± standard deviation (SD) and are significant at * p <0.05 and ** p <0.01 by independent sample t-test. ^#: TG (Triglyceride), CE (cholesterol esters), FC (free cholesterol) were both quantitative analysis using the above-described HPLC chromatography with standard gokseonreul of TG, CE and FC. ^& : CE / FC is the mean standard deviation for each ratio. ^$ : KU is the Karmen unit.

(3) biochemical analysis of feces

In contrast to the decrease in total lipid, TG and TC contents in serum and liver, the total amount of lipids, TG and TC in excreta were all increased in specific amounts, total lipid secretion increased by 21-47% and TG increased by 17-78%, respectively. Increased 0-126% (Table 4). Among them, the change in the amount of total cholesterol was found to be concentration dependent, which gradually increased with increasing dose.

Dung levels after 8 weeks of Platicodin D (PD) administration Parameter 15 mg / kg PD 30mg / kg PD 50 mg / kg PD Control Normal Diet Method HC HC HC HC HC-free ^a total lipid extract 12.2 ± 1.9 12.0 ± 1.3 * 14.6 ± 3.0 * 9.9 ± 1.8 6.3 ± 2.1 TG 7.3 ± 3.0 5.1 ± 1.4 4.8 ± 1.5 4.1 ± 0.7 3.1 ± 1.2 ^b cholesterol 1.3 ± 0.2 1.8 ± 1.0 3.4 ± 1.2 1.5 ± 0.4 1.1 ± 0.7 The figures are expressed as mean ± standard deviation (SD) and are significant at * p <0.05 and ** p <0.01 by independent sample t-test. ^a : The total lipid extract was extracted using Soxhlet lipid extraction. ^b : Cholesterol was measured using a commercially available kit and included some cholesterol metabolites that remained as cholesterol esters, free cholesterol and un-oxidized 3-hydroxyl residues.

Experimental Example 3: Effect of Platicodin D on Factors of Atheromatous Atheroogenesis

In vitro (in in vitro Inhibitory Effect of Platicodin D on ACAT

In vitro inhibitory activity against ACAT-1 or ACAT-2 was measured according to known methods using expressed human ACAT-1 or -2 obtained from Hi5 cells as an enzyme source (Cho KH, Lee AS, Paik WS, Kim YK, and Jeong YK, Mass-production of human ACAT-1 and ACAT-2 to screen isoform-specific inhibitors: a different substrate specificity and inhibitory regulation.Biochem Biophys Res Commun 309: 864-872,2003). Microsome fractions were obtained by microcentrifugation with a Beckman L8-M microcentrifuge (Palo Alto, CA) using a SW55.1 rotor.

As shown in FIG. 2, the inhibitory activity was 42% and 41% for human ACAT-1 and ACAT-2, respectively, when the Platicodin D final concentration was 81 μM. Isotypes of ACAT are known to act differently in terms of many biochemical and biophysical properties such as substrate binding efficiency, membrane topology and dislocation of tissue locations. Therefore, simultaneous inhibition of ACAT-1 and ACAT-2 by Platicodin D may maximize the therapeutic efficiency of Platicodin D to lower cholesterol levels.

Binding Activity of Platicodin D and Farnesoid X Receptor (FXR) in Vitro

Co-activator recruitment analysis for FXR activity was performed using recombinant FXR protein and its natural ligand, chenodeoxycholic acid (CDCA). FXR activation was measured using an optical microplate reader (Wallac Victor2, Perkin-Elmer) with the addition of 1 nM of DELFIA-Eu-N1 labeled anti-His antibody (Perkin-Elmer, Foster City, Calif.). (Kelli S, Bramlett SY, and Thomas PB, Correlation of Farnesoid X receptor coactivator recruitment and cholesterol 7-hydroxylase gene repression by bile acids.Mol Gene Metab, 71: 609-61, 2000). The Platicodin D fraction was dissolved in 50% ethanol and various Platicodine D fractions were prepared in 3 ml doses.

Platicodin D was determined to antagonize the nuclear FXR (FIG. 3). Platicodin D reduced FXR activity promoted by CDCA, a natural ligand of FXR. Treatment with Platicodin D at a final concentration of 40 μM showed an inhibitory effect of 40-50% on CDCA control levels.

Experimental Example 4: In vivo (in vivo Measurement of Polymer Formation between Platicodin D and Cholesterol

Cholesterol standard solution (3 mg / ml-7.75 mM) of the cholesterol analysis kit (Eiken Chemicals, Japan) was used as a reference. The same amount of cholesterol solution was added to the Platicodin D solution by concentration. The mixed solution was reacted for 3 hours at room temperature with shaking, and the cholesterol concentration in the suspension was measured using the same assay kit described above, and then the precipitate was removed by centrifugation. Triglyceride (TG) standard solution (3 mg / ml) was used as a control. Other platicodine saponin compounds were also used to compare the polymer forming ability with cholesterol.

Platicodine D has been found to form insoluble polymers with cholesterol, and FIG. 4 shows a stoichiometric platicodine concentration of 8.3 mg / ml (≒ 6.77 mM) with 50% cholesterol precipitation. It was shown that the ratio of D and cholesterol was found to be about 1: 1. In contrast, Platicodin D did not form a polymer with TG. Polymer formation was also shown to be related to the polarity of platicodine saponin (PS). For example, polygalacin D and placecodin A, which have a lower polarity than placodine D, exhibit a strong affinity for cholesterol, while another PS, which has a high polarity, platicodine D ₃ hardly forms polymers. Did.

Examples of preparations for the compositions of the present invention are illustrated below.

Formulation Example 1 Preparation of Pharmaceutical Formulation

1. Preparation of powder

Platicodine D 2g

1g lactose

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

2. Preparation of Tablets

Platicodine D 100mg

Corn Starch 100mg

Lactose 100mg

2 mg magnesium stearate

After mixing the above components, tablets were prepared by tableting according to a conventional method for producing tablets.

3. Preparation of Capsule

Platicodine D 100mg

Corn Starch 100mg

Lactose 100mg

2 mg magnesium stearate

After mixing the above components, the capsule was prepared by filling in gelatin capsules according to the conventional method for producing a capsule.

Formulation Example 2: Preparation of Food

Foods containing Platicodin D of the present invention were prepared as follows.

1. Preparation of flour food

Platicodin D 0.5 to 5.0% by weight of the present invention was added to the flour, and using this mixture to prepare bread, cakes, cookies, crackers and noodles to prepare health foods.

2. Manufacturing of Dairy Products

Platycodine D 5-10% by weight of the present invention was added to milk, and the milk was used to prepare various dairy products such as butter and ice cream.

3. Manufacture of wire

Brown rice, barley, glutinous rice, and yulmu were alphad by a known method, and then dried and roasted to prepare a powder having a particle size of 60 mesh using a grinder.

Black beans, black sesame seeds, and perilla were also steamed and dried by a known method, and then ground to a powder having a particle size of 60 mesh.

Platicodine D of the present invention was concentrated under reduced pressure in a vacuum concentrator, and the dried product obtained by drying with a sprayer and a hot air dryer was pulverized with a particle size of 60 mesh to obtain a dry powder.

The grains, seeds and the dry powder of Platicodin D prepared above were blended in the following ratios.

Cereals (30% by weight brown rice, 15% by weight radish, 20% by weight barley),

Seeds (7% by weight perilla, 8% by weight black beans, 7% by weight black sesame),

Dry powder of Platicodin D (3% by weight),

Ganoderma lucidum (0.5% by weight),

Foxglove (0.5 wt%)

Formulation Example 3: Preparation of Beverage

1. Manufacture of health drinks

Immediately sterilize by homogeneously mixing Platicodine D with subsidiary materials such as liquid fructose (0.5%), oligosaccharide (2%), sugar (2%), salt (0.5%) and water (75%). Healthy drinks were prepared by packing them in small packaging containers such as bottles and plastic bottles.

2. Preparation of Vegetable Juice

5 g of Platicodin D of the present invention was added to 1,000 ml of tomato or carrot juice to prepare vegetable juice for health promotion.

3. Preparation of Fruit Juice

1 g of Platicodine D of the present invention was added to 1,000 ml of apple or grape juice to prepare fruit juice for health promotion.

As discussed above, the composition comprising placodin D of the present invention as an active ingredient reduces the weight and food intake of the individual, and serves to reduce low density cholesterol (LDL-C) in the blood, liver and excreta. . More specifically, by inhibiting acyl coenzyme A: cholesterol acyltransferase (ACAT), which plays an important role in atherosclerotic lesionogenesis in the artery, and by antagonizing the farnesoid X receptor (FXR) It has the effect of reducing the risk of atheromatous atherosclerosis in the artery. Formation of cholesterol and insoluble polymers inhibits the absorption of cholesterol. It can be usefully used for the prevention and treatment of cholesterol-related diseases such as peripheral vascular diseases.

Claims (8)

A prophylactic and therapeutic agent for hypercholesterolemia containing Platicodin D as an active ingredient. The method for preventing and treating hypercholesterolosis according to claim 1, wherein the prophylactic and therapeutic agent is used for the prevention and treatment of obesity. The method of claim 1, wherein the prophylactic and therapeutic agents are used for the prevention and treatment of diseases selected from the group consisting of hyperlipidemia, arteriosclerosis, hypertension, myocardial infarction, angina pectoris, stroke, cerebral hemorrhage, peripheral vascular disease, and the like. Prevention and treatment of cholesterol. ACAT (acyl-coenzyme A: cholesterol acyltransferase) activity inhibitor containing Platicodin D as an active ingredient. Farnesoid X Receptor antagonist containing Platicodin D as an active ingredient.  Inhibitor of atheromatous lesion formation in the intima of the artery containing Platicodin D as an active ingredient. The method for preventing and treating hypercholesterolosis according to claim 1, wherein the prophylactic and therapeutic agent is a pharmaceutical composition.  The method for preventing and treating hypercholesterolosis according to claim 1, wherein the prophylactic and therapeutic agent is a health food.

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