KR100552109B1 - Composition for preventing and treating cardiovascular disease containing sesquiterpene lactone extracted from Ixeris dentata forma albiflora - Google Patents

Abstract

The present invention relates to a composition for treating cardiovascular disease and cancer containing a sesquiterpene lactone compound extracted from white bark. Zaluzanin C, 9α-hydroxyguaian-4 (15), 10 (14), 11 (13) -triene-6,12-olide and Eryrin M, which are sesquiterpene lactone compounds extracted from white bark Has ACAT inhibitory activity, DGAT inhibitory activity and FPTase inhibitory activity. Thus, the compounds can be used for the prevention and treatment of cardiovascular disease and cancer.

Whitefish, Sesquiterpene Lactone, Cardiovascular Disease, Cancer

Description

Composition for preventing and treating cardiovascular disease containing sesquiterpene lactone extracted from Ixeris dentata forma albiflora}

Figure 1 shows the results of nuclear magnetic resonance (NMR) spectrum analysis of zaluzanin C, a sesquiterpene lactone compound extracted from the root of a white muscus.

Figure 2 shows the infrared (IR) spectral analysis of the zaluzanin C (seluquitin pen lactone compound extracted from the root of the white mudfish).

3 is a sesquiterpene lactone compound 9α-hydroxyguan-4 (15), 10 (14), 11 (13) -triene-6,12-oxide (9α- Nuclear Magnetic Resonance (NMR) spectra of hydroxyguaian-4 (15), 10 (14), 11 (13) -triene-6,12-olide are shown.

4 is a sesquiterpene lactone compound 9α-hydroxyguan-4 (15), 10 (14), 11 (13) -triene-6,12-oxide (9α- Infrared (IR) spectrum analysis results for hydroxyguaian-4 (15), 10 (14), 11 (13) -triene-6,12-olide) are shown.

Figure 5 shows the results of nuclear magnetic resonance (NMR) spectrum analysis of the acetylide of the sesquiterpene lactone compound ixerin M extracted from the rhizome of the white mudfish.

Figure 6 shows the infrared (IR) spectral analysis of the acetylide of the sesquiterpene lactone compound ixerin M extracted from the rhizome of the white moth.

The present invention relates to a composition containing a sesquiterpene lactone compound, and more particularly, to a composition for treating cardiovascular disease and cancer containing a sesquiterpene lactone compound extracted from a white mucus.

Cardiovascular disease is known to be the leading cause of death in the United States and Europe, and is one of the most frequent diseases among Koreans. Factors causing cardiovascular disease include hereditary predisposition, sex, smoking, lifestyle and age. In particular, hypercholesterolemia caused by low density lipoprotein cholesterol (hereinafter referred to as 'LDL-cholesterol') and total cholesterol is a major cause of cardiovascular disease. Thus, lowering blood LDL-cholesterol and total cholesterol levels can reduce the incidence of cardiovascular diseases such as atherosclerosis and myocardial infarction.

Cholesterol lowering agents used to lower blood LDL-cholesterol and total cholesterol levels. Nicotinic acid, cholestyramine, clofibrate (CPIB), neomycin, and plant sterols. Drugs such as triparanol (MER-29), D-thyroxine and estrogen have been used. However, many problems have been reported with these drugs. That is, it is known to cause side effects such as skin vasodilation, rash, gastrointestinal abnormality, uric acidemia, hyperglycemia, liver dysfunction, or causing an unpleasant feeling due to an overdose. Recently, inhibitors that inhibit the activity of HMG-CoA reductase, such as lovastatin, have been developed and used as cholesterol lowering agents (Hunninghake, Curr. Opin. Lipidol ., 3: 22-28, 1992; Brown et al ., N. Engl. J. Med ., 323: 1289-1298, 1990). However, inhibitors of HMG-CoA reductase activity, such as lovastatin, have potent inhibitory effects, such as myopathy, rhabdomyolysis, suicidal tendency, neurosis, and neurosis. It is known to cause side effects (Corpier et al ., JAMA , 260: 239-241, 1988; Pierce et al ., JAMA , 264: 71-75, 1990; Reaven and Witztum, Ann.Intern. Med ., 109: 597-598, 1988; Duits and Bos, Lancet , 341: 114, 1993; Engelberg, Lancet , 339: 727-729, 1992). Therefore, there is an increasing demand for a new type of cardiovascular disease therapeutic agent that can supplement the problems of existing therapeutic agents without side effects.

Meanwhile, cancer is also a disease that frequently occurs in the US, Europe, and Korea, and about 50 kinds of anticancer drugs are used. However, since these anticancer drugs act on all cells that divide or proliferate rapidly, they also damage cells that are normally active in cell division (bone marrow cells, gastrointestinal epithelial cells, hair follicles), but there are side effects such as bone marrow suppression, gastrointestinal disorders, and hair loss. It is known to occur in almost all patients. Therefore, there is a need for development of a new type of anticancer agent that can supplement the problems of existing therapeutic agents without side effects.

Sesquiterpenoids (sesquiterpenoid) is a kind of terpenoid compound (terpenoid), widely distributed in a variety of plants, and more than 100 kinds of basic skeleton is known. Of these sesquiterpenoid compounds, in particular, sesquiterpene lactone having a lactone structure is one of a group of plant natural compounds in which various physiological activities have been reported. The physiological activity of this compound group is known as anti-tumor activity, cytotoxicity, antimicrobial action and insecticidal action, and pharmacological activity such as gastrointestinal cell protection or analgesic action has also been reported.

Asteraceae plants are known to contain most of these sesquiterpene lactone compounds, and costunolide, one of the sesquiterpene lactone compounds, has been isolated from the neck (Kang et al ., Kor. J. Pharmacogn . , 30: 137-144, 1999), in particular, hemistepsin A / S isolated from gujeolcho and mountain countries, angelerolcumambrin B, cumambrin A / B), sesquiterpene lactone compounds such as trigloylcumambrin B, tullphinolide, costunolide, and handelin inhibit both ACAT and FPTase activity (Yang et al ., Kor. J. Pharmacogn ., 29 (3): 243-247, 1998; Jang et al ., Kor. J. Pharmacogn ., 30 (1): 74). -78, 1999; Jang et al ., Kor. J. Pharmacogn ., 30 (1): 70-73, 1999; Khalil et al ., Planta Medica ., 57: 190, 1991). However, studies on the specific function of hundreds of sesquiterpene lactone compounds are still insufficient.

Ixeris dentata NAKAI is a perennial herb belonging to the Asteraceae family and is known to contain more than 80 kinds of volatile flavor essential oils, including hexenol. These writings are not only inferior in edible aspect, but also occupy an important part in the Korean diet because of their unique and diverse flavors. In addition, the civilian has been used for a long time in tonic protection, anorexia, gastritis, sedation, pain, sinusitis, fever, etc., and recently, it is known that the anticancer effect of inhibiting human bone cancer cells and the effect of lowering cholesterol are great. In addition, it has been reported that there is an effect on antioxidant, antistress, antibacterial, immune enhancement, intestinal function recovery, weight control (Korean Patent Application No. 2001- 3744).

As a result, the chemicals contain a large amount of vitamin A, polysaccharides, inulin, alcohols, esters, aldehydes, ketones, hydrocarbons, acids, phenols, flavonoids, and guanes Sesquis. It has been reported to contain terpene lactone compound glycoside components (Kim et al ., J. Korean Agric. Chem. Soc. , 31 (4): 394-399, 1988; Chung et al ., Arch. Pharm. Res . , 17 (5): 327, 1994). Yellow flowers and white flowers are called Ixeris dentata forma albiflora . However, there are few studies on the chemical analysis, physiological activity and clinical effects of white flowers.

The inventors of the present invention, while reducing the blood cholesterol level using one of the natural herbal medicines, while researching to find a biologically active substance having anti-cancer activity, from a long-term natural herbal medicine that has been used for a long time Sesquiterpene lactone compounds were isolated and purified, and the present invention was completed by confirming that the compounds had ACAT inhibitory activity, DGAT inhibitory activity, and FPTase inhibitory activity, and thus could be used as therapeutic agents for cardiovascular diseases and cancer.

The technical problem to be achieved by the present invention is to provide a composition containing a sesquiterpene lactone compound that can be used as a therapeutic agent for cardiovascular diseases and cancer.

In order to achieve the above object, the present invention is a zaluzanin C (zaluzanin C) of the formula (1), 9α-hydroxyguaian-4 (15), 10 (14), 11 (13) Triene-6,12-oxide (9α-hydroxyguaian-4 (15), 10 (14), 11 (13) -triene-6,12-olide) and Eryrin M (ixerin M) It provides a composition for preventing and treating cardiovascular diseases containing at least one compound selected from the group consisting of.

In addition, the present invention is a zaluzanin C of formula (1), 9α-hydroxyguan-4 (15), 10 (14), 11 (13) -triene-6, 12- of formula (2) At least one compound selected from the group consisting of (9α-hydroxyguaian-4 (15), 10 (14), 11 (13) -triene-6,12-olide) and ixerin M of Formula 3 It provides a composition for preventing and treating cancer containing.

<Formula 1>

<Formula 2>

<Formula 3>

Hereinafter, the present invention will be described in detail.

In the present invention, sesquiterpene lactone compound, zaluzanin C, 9α-hydroxyguan-4 (15), 10 (14), 11 (13) -triene-6,12- Cardiovascular vessels containing one or more compounds selected from the group consisting of old (9α-hydroxyguaian-4 (15), 10 (14), 11 (13) -triene-6,12-olide) and ixerin M It is characterized by providing a composition for preventing and treating diseases.

The sesquiterpene lactone compound according to the present invention may be preferably used in a composition for preventing and treating cardiovascular diseases such as atherosclerosis, hypertension, angina pectoris, hyperlipidemia, myocardial infarction or heart failure (Carlson, LA et al ., Acta Med. Scan ., 206: 351, 1979).

Zaluzanin C, 9α-hydroxyguaian-4 (15), 10 (14), 11 (13) -triene-6,12-oxide (9α-hydroxyguaian-4 (15), 10 (14), 11 (13) -triene-6,12-olide) and ixerin M may be extracted from Ixeris dentata forma albiflora according to conventional methods known in the art. Preferably, the root root of the white bark can be extracted with lower alcohol. Specifically, the roots of the white bark extract were extracted with 80% methanol, partitioned extraction using ethyl acetate, n-butanol, and water, and the resulting fractions were concentrated under reduced pressure to obtain silica gel column chromatography. Can be. In this case, as the developing solvent, various organic solvents known in the art may be used. Preferably, a mixed solvent of n-hexane and ethyl acetate and a mixed solvent of chloroform and methanol may be used.

In one embodiment of the present invention, the ACAT activity inhibition measurement method and DGAT activity inhibition measurement method were used to determine whether the sesquiterpene lactone compound extracted from the white bark can be used as a cardiovascular agent.

Acyl CoA: cholesterol acyltransferase (ACAT) is one of the target enzymes for the development of new cardiovascular disease therapies with definite mechanisms of action and no side effects. ACAT promotes the absorption of cholesterol into the small intestine by transforming cholesterol into the form of cholesterol esters (CE), and 2) forming very low-density lipoproteins (VLDL) in the liver. It is known to be involved in the formation of low-density lipoprotein (LDL), which is directly related and the decisive factor in atherosclerosis, and 3) is also involved in the accumulation of CE in the surrounding cells of vascular wall wounds. In addition, the presence of more than CE is known to accumulate in the blood vessel wall or increase the viscosity of the blood, causing various cardiovascular diseases. Therefore, by inhibiting the activity of ACAT, it is possible to lower the viscosity of the blood by lowering the amount of CE in the blood, to reduce the synthesis of LDL by inhibiting the formation of VLDL in the liver, the amount of cholesterol absorbed by the intestine As a result, it will be able to treat various cardiovascular diseases.

In addition, DGAT (Acyl CoA: diacylglycerol acyltransferase) is an important enzyme involved in the biosynthesis of triglycerides. DGAT is involved in: 1) absorption of fatty acids in the small intestine, 2) control of triglyceride levels in the blood, 3) fat storage in adipocytes, 4) energy metabolism and milk production in muscles, and in the case of plants, (Lassner et al ., Lipid Technol ., 9: 5-9, 1997). Triacylglycerol is known to play an important role in energy storage. When it accumulates excessively in tissues and organs of the body, it causes fatty liver, obesity, hypertriglyceridemia, etc. It causes serious diseases such as metabolic disorders and deterioration of organs. Therefore, if you inhibit the activity of DGAT to inhibit triglyceride biosynthesis will be able to treat a variety of cardiovascular diseases.

On the other hand, in the present invention, sesquiterpene lactone compounds (zaluzanin C), 9α-hydroxyguan-4 (15), 10 (14), 11 (13) -triene-6, Contains at least one compound selected from the group consisting of 12-oxides (9α-hydroxyguaian-4 (15), 10 (14), 11 (13) -triene-6,12-olide) and ixerin M It is characterized by providing a composition for preventing and treating cancer.

In one embodiment of the present invention was used to measure the inhibition of FPTase activity in order to determine whether the sesquiterpene lactone compound extracted from the white bark can be used as a cancer treatment. Farnesyl-protein transferase (FPTase) has been evaluated as a useful target enzyme for the development of effective cancer therapeutics. This FPTase is one of the enzymes involved in the signaling process that regulates cell proliferation and differentiation. It is responsible for delivering farnesyl groups of farnesyl pyrophosphate to ras protein. (Pompliano et al ., Biochem ., 31: 3800-3807, 1992). Ras proteins are the most important proteins in signal transduction through growth regulators and are expressed by the ras proto-oncogene, which is known to be associated with malignant tumors (Lowy et. al ., Ann. Rev. Biochem ., 62: 851-891, 1993). If this FPTase enzyme can be inhibited, cancer may be effectively treated. Therefore, the compounds of the present invention having the activity of inhibiting the FPTase enzyme can be used for the prevention and treatment of cancer, and specifically related to the las protein such as pancreatic cancer, bladder cancer, lung cancer or skin cancer known to be related to the las protein. It can be used for the prevention and treatment of cancer known to be known (Bos, JL, Cancer Res ., 49: 4682, 1989).

Zaluzanin C, 9α-hydroxyguaian-4 (15), 10 (14), 11 (13) -triene-6,12-oxide, which are sesquiterpene lactone compounds according to the present invention 9α-hydroxyguaian-4 (15), 10 (14), 11 (13) -triene-6,12-olide) and ixerin M are suitable carriers or excipients according to conventional methods known in the art. It may be mixed with or diluted with a diluent to prepare a composition for treating cardiovascular diseases and cancer.

Examples of suitable carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, undetermined Vaginal cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil and the like. The composition may further include fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives and the like.

Compositions of the present invention can be formulated using methods known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal. The formulations may be in the form of tablets, pills, powders, sachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols, soft or hard gelatin capsules, sterile injectable solutions, sterile powders and the like.

The compositions of the present invention can be administered via several routes including oral, transdermal, subcutaneous, intravenous or intramuscular. For humans a typical daily dosage of the active compound may range from 1 to 1,000 mg, preferably 10 to 100 mg per kg of body weight, and may be administered once or in divided doses. However, it should be understood that the actual dosage of the active ingredient should be determined in light of several relevant factors such as the disease to be treated, the route of administration, the age, sex, weight of the patient and the severity of the disease, and therefore the dosage may be determined in some way. Nor does it limit the scope of the invention.

On the other hand, the composition of the present invention may include a pharmaceutically acceptable salt.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

<Example 1>

Isolation and Purification of Sesquiterpene Lactone Compounds from White Root Root

White moth used in the present invention ( Ixeris dentate forma albiflora ) was purchased in October 2002 from Yangpyeong-gun, Yangpyeong-gun, Gyeonggi-do. First, 60 kg of white-tailed shellfish were divided into the underground part (root 45%) and the ground part (55% leaf), and the underground part was cut to about 2cm in size.

27 kg of the roots of the white mud was immersed in 360 L of 80% aqueous methanol solution for 48 hours and extracted at room temperature. The extract was filtered and the remaining one was extracted twice more in the same way. All the obtained filtrates were combined, and it concentrated under reduced pressure at 45 degreeC by the vacuum concentrator. The methanol extract was partitioned and extracted with a mixture of 2 liters of ethyl acetate and 2 liters of water, and the aqueous layer was partitioned and extracted with a mixture of 2 liters of n-butanol and 2 liters of water. Each layer was concentrated under reduced pressure to obtain 17 g of ethyl acetate fraction, 80 g of n-butanol fraction, and 251 g of water fraction.

After dipping into 10% aqueous H ₂ SO ₄ and UV absorption on TLC (thin layer chromatography), the ethyl acetate fraction and n-butanol fraction were estimated to contain the active substance by color development. Silica gel column chromatography was performed.

1-1) Separation of Sesquiterpene Lactone Compounds 1 and 2 from Ethyl Acetate Fraction

Silica gel column chromatography was performed on 17 g of the ethyl acetate fraction, and a mixed solvent of n-hexane and ethyl acetate and a mixed solvent of chloroform and methanol were used as a developing solvent. The ratio of n-hexane to ethyl acetate was 7: 1, 3: 1, 1: 1, and the ratio of chloroform and methanol was 5: 1, 2: 1. Then, each fraction obtained was identified by TLC, and similar portions were collected and concentrated to obtain eight fractions. At this time, a mixed solvent of n-hexane and ethyl acetate was used in a ratio of 5: 1 and 2: 1, and a mixed solvent of chloroform and methanol was used in a ratio of 5: 1 and 2: 1. The eight fractions were subjected to silica gel column chromatography using chloroform and methanol (10: 1, 7: 1, 5: 1) as elution solvents to obtain 6 fractions. Was purified by silica gel column chromatography using n-hexane and ethyl acetate (3: 1) as the elution solvent to finally separate and purify the active fractions of Compound 1 and Compound 2 in amounts of 20 mg and 15 mg, respectively.

1-2) Separation of Sesquiterpene Lactone Compound 3 from n-butanol Fraction

Silica gel column chromatography was performed on 80 g of the n-butanol fraction, and a mixed solvent of chloroform, methanol, and water was used as a developing solvent. The ratio of chloroform, methanol, and water was 9: 3: 1, 7: 3: 1, 65:35:10. Thereafter, each fraction was identified by TLC, and similar ones were collected and divided into 10 small fractions. Silica gel column chromatography was again performed on one of the ten fractions (n-hexane: chloroform: methanol = 2: 3: 1) and divided into five subfractions. Acetylation was carried out by adding 3 ml of pyridine and the same amount of acetic anhydride under ice-cooling to purify the compound more purely and to facilitate structural analysis of the third fraction. And stirred for one day at room temperature. The acetylated fractions were partitioned between ethyl acetate and water and extracted. The organic layer was washed with 5% HCl, saturated NaHCO ₃ and NaCl. This was dehydrated with anhydrous MgSO ₄ , filtered and concentrated under reduced pressure. This was further subjected to silica gel column chromatography using a solvent of n-hexane: ethyl acetate (1: 1) to separate and purify the acetylated compound 3a of the active fraction 3 in an amount of 22.4 mg.

<Example 2>

Analysis of Physicochemical Properties of Compounds Isolated and Purified from White Clover

2-1) Physical and Chemical Properties of Compound 1

Compound 1 had a [α] _D value of + 2.9 ° and was developed on TLC to show no UV absorption. In addition, when spray-dried and heated with 10% H ₂ SO ₄ , it developed a purple color. The compound 1 was completely dried, dissolved in CDCl ₃ , and placed in a 5 mm tube, and subjected to NMR analysis using a nuclear magnetic resonance spectrometer (Nuclear Magnetic Resonance spectrometer, Varian Inova AS 400, Varian, USA). ¹ H-NMR (400 MHz, CDCl ₃ ) and DEPT (Distortionless Enhancement by Polarization Transfer) & ¹³ C-NMR (100 MHz, CDCl ₃ ) results are shown in Table 1 and FIG. 1.

In addition, the compound 1 was dissolved in chloroform and dried, and then analyzed by infrared (IR) absorbance using an infrared spectrometer (Infrared Spectrometer, Perkin model 599B, Perkin-Elmer, Massachusetts, USA). IR absorbance showed peaks of 3436 (broad, -OH), 2967, 1765 (C = O) and 135 cm ^-1 (KBr) (see FIG. 2).

As a result of the analysis, Compound 1 has a lactone ring, and it can be confirmed that it is zaluzanin C, a sesquiterpene lactone compound having one hydroxyl group and three exomethylene. (Otmar, Spring. Et al ., Phytochemistry , 39: 609-612, 1995).

Carbon number δ _H δ _C One ^{2.91 (1 H, br, dd} , J = 7.6, 8.4 Hz) 44.36 2a 2.32 ⁽¹ H, m) 39.19 2b 1.75 ⁽¹ H, m) 3 ^{4.58 (1 H, dddd, J} = 7.6, 2.0, 2.0, 2.0 Hz) 73.73 4 153.19 5 2.86 ( ¹ H, dd, J = 9.6, 7.6 Hz) 50.09 6 4.11 ( ¹ H, doublet of doublets, J = 9.2, 9.6 Hz) 84.15 7 2.84 ⁽¹ H, m) 45.77 8a 2.25 ⁽¹ H, m) 30.77 8b ^{1.46 (1 H, br. S} ) 9a 2.48 ( ¹ H, dd, J = 6.0, 6.8 Hz) 34.37 9b 2,17 ⁽¹ H, m) 10 148.15 11 139.85 12 170.36 13a 6.20 ( ¹ H, d, J = 3.4 Hz) 120.51 13b 5.50 ( ¹ H, d, J = 3.4 Hz) 14a ^{5.01 (1 H, d, J} = 1.2 Hz) 114.62 14b 4.95 ( ¹ H, d, J = 1.2 Hz) 15a 5.45 ( ¹ H, dd, J = 2.0, 2.0 Hz) 111.45 15b ^{5.33 (1 H, dd, J} = 2.0, 2.0 Hz)

2-2) Physical And Chemical Properties Of Compound 2

Compound 2 had a [α] _D value of + 0.3 ° and developed by TLC, and showed no UV absorption. In addition, when spray-dried and heated with 10% H ₂ SO ₄ , it developed a purple color. Compound 2 was completely dried, dissolved in CDCl ₃ , and placed in a 5 mm tube to perform nuclear magnetic resonance analysis (NMR). ¹ H-NMR (400 MHz, CDCl ₃ ) and DEPT & ¹³ C-NMR (100 MHz, CDCl ₃ ) results are shown in Table 2 and FIG. 3.

Further, Compound 2 was dissolved in chloroform and dried, and then infrared (IR) absorbance was analyzed using an infrared spectrometer. IR absorbance showed peaks of 3460 (-OH), 1766 (C = O), and 1320 cm ^-1 (KBr) (see FIG. 4).

As a result of the analysis, the compound 2 is a compound of the same guaninolide series as compound 1, but 9α-hydroxyguaian-4 (15), 10 (14) which is a compound substituted with a hydroxyl group at carbon 9 ), 11 (13) -triene-6,12-oxide (9α-hydroxyguaian-4 (15), 10 (14), 11 (13) -triene-6,12-olide) was confirmed (J , Y. et al ., Phytochemistry , 48: 201-203, 1998).

Carbon number δ _H δ _C One ^{3.42 (1 H, ddd, J} = 8.0, 8.4, 2.4 Hz) 38.84 2a 1.97 ( ¹ H, m) 30.06 2b 1.80 ( ¹ H, m) 3a 2.53 ( ¹ H, m) 33.23 3b 2.17 ⁽¹ H, m) 4 151.59 5 2.88 ⁽¹ H, m) 51.78 6 3.86 ( ¹ H, doublet of doublets, J = 9.2, 9.2 Hz) 86.19 7 3.20 ⁽¹ H, m) 40.39 8a 2.28 ⁽¹ H, m) 39.20 8b 1.58 ( ¹ H, m) 9 ^{4.53 (1 H, dd, J} = 3.6, 3.6 Hz) 74.22 10 151.50 11 139.42 12 170.69 13a 6.24 ( ¹ H, d, J = 3.4 Hz) 120.63 13b 5.52 ( ¹ H, d, J = 3.4 Hz) 14a ^{5.03 (1 H, br. S} ) 112.91 14b ^{4.83 (1 H, br. S} ) 15a ^{5.22 (1 H, ddd, J} = 2.0, 2.0, 2.0 Hz) 109.27 15b ^{5.03 (1 H, br. S} )

2-3) Physical and Chemical Properties of Compound 3a

Compound 3a had a [α] _D value of ?? 0.6 ° and was observed on TLC to show no UV absorption. In addition, when spray-dried and heated with 10% H ₂ SO ₄ , it developed a purple color. Compound 3a was completely dried, dissolved in CDCl ₃ , and placed in a 5 mm tube to perform nuclear magnetic resonance analysis (NMR). ¹ H-NMR (400 MHz, CDCl ₃ ) and DEPT & ¹³ C-NMR (100 MHz, CDCl ₃ ) results are shown in Table 3 and FIG. 5.

Further, Compound 3a was dissolved in chloroform and dried, and then infrared (IR) absorbance was analyzed using an infrared spectrometer. IR absorbance showed peaks of 3420 (-OH), 1670 (C = O), and 1220 cm ^-1 (KBr) (see FIG. 6).

As a result of the analysis, the compound 3a is a D-glucose molecule and 2-hydroxy in the sesquiterpene lactone structure in which three exomethylene and three oxygenated methine carbons are present It was confirmed that it is an acetylate of erythrine M (ixerin M), a compound having a 3-hydroxybutynoyl (2-hydroxy-3-methylbutanoyl) group (T, W. et al ., Phytochemistry , 29: 3217-3224, 1990). In the following activity experiment, Compound 3, in which the acetyl group was removed from Compound 3a, was used.

Carbon number δ _H δ _C One ^{2.92 (1 H, br. Dd} , J = 5.3, 10.8 Hz) 45.51 2a 2.34 ( ¹ H, m) 37.97 2b 1.98 ( ¹ H, m) 3 ^{4.52 (1 H, br. S} ) 81.22 4 149.12 5 ^{2.74 (1 H, dd, J} = 9.5, 6.3 Hz) 51.44 6 ^{4.44 (1 H, dd, J} = 6.4, 5.9 Hz) 78.48 7 ^{3.14 (1 H, dd, J} = 5.9, 1.8 Hz) 48.25 8 ^{5.60 (1 H, br. S} ) 68.71 9a 2.43 ( ¹ H, dd, J = 13.9, 6.1 Hz) 39.64 9b 2.42 ( ¹ H, dd, J = 13.9, 5.8 Hz) 10 142.17 11 134.39 12 170.90 13a 6.33 ( ¹ H, d, J = 2.0 Hz) 123.31 13b ^{5.38 (1 H, d, J} = 2.0 Hz) 14a ^{5.13 (1 H, br. S} ) 118.74 14b ^{4.95 (1 H, br. S} ) 15a ^{5.56 (1 H, br. S} ) 113.92 15b ^{5.25 (1 H, br. S} )   One' ^{4.74 (1 H, d, J} = 8.0 Hz) 100.79   2' ^{5.09 (1 H, dd, J} = 9.7, 8.0 Hz) 71.76   3 ′ ^{5.24 (1 H, dd, J} = 9.5, 9.0 Hz) 73.31   4' ^{5.05 (1 H, dd, J} = 9.1, 9.5 Hz) 69.02   5 ′ 3.73 ⁽¹ H, m) 72.18   6a ′ ^{4.26 (1 H, dd, J} = 12.1, 4.8 Hz) 62.50   6b ′ ^{4.16 (1 H, br. D} , J = 9.7, 8.0 Hz) α 169.81 β 4.72 ( ¹ H, d, J = 4.3 Hz) 77.05 γ 2.17 ⁽¹ H, m) 30.13 δ ^{1.00 (3 H, d, J} = 6.8 Hz) 19.16   δ ′ ^{0.96 (3 H, d, J} = 6.8 Hz) 17.47 Acetyl ^{2.11 (3 H, s),} 2.04 (3 H, s) 2.08 (3 H, s), 2.02 (3 H, s), 2.01 (3 H, s) 170.98, 170.96 170.52, 169.56, 169.45, 21.08, 20.95 × 2, 20.91, 20.86

<Example 3>

Determination of ACAT Enzyme Inhibitory Activity and DGAT Enzyme Inhibitory Activity of Sesquiterpene Compounds

3-1) Determination of ACAT Enzyme Activity Inhibition of Sesquiterpene Compounds

ACAT enzyme inhibitory activity of each sesquiterpene lactone compound was measured in order to confirm whether the sesquiterpene lactone compound isolated and purified from white bark can be used to treat cardiovascular disease.

ACAT activity measurement was performed by Tabas et al . (Tabas et al ., J. Biol. Chem ., 261: 3147-3154) using [ ^1-14 C] oleoyl-CoA as a substrate. , 1986). 10 μl of the compound sample solution, 4 μl of rat liver microsomal enzyme, 20 μl of assay buffer (0.5M KH ₂ PO ₄ , 10 mM DTT, pH 7.4), 40 mg / ml BSA (essentially fatty) acid free) 15 µl, 20 µg / ml cholesterol, 2 µl and 41 µl of distilled water were preliminarily reacted at 37 ° C. for 20 minutes, and then [ ^1-14 C] oleoyl-CoA was added to the reaction solution. 8 μl (0.05 μCi, final concentration 10 μM) was added and the reaction was carried out again at 37 ° C. for 25 minutes. Thereafter, 1 ml of isopropanol-heptane (4: 1, v / v) was added to stop the reaction. 0.6 ml of heptane and 0.4 ml of a 5-fold diluted assay buffer were added, followed by centrifugation. Enzyme activity was measured by adding 3 ml of scintillation cocktail to 100 μl of the supernatant obtained by centrifugation, and then measuring the radioactivity using a liquid scintillation counter.

ACAT inhibition was measured by using a radioactivity measuring device to measure the amount of the reaction product produced by adding a test sample to a mixture of the substrate and enzyme labeled with radioactivity, and the inhibition was calculated by the following equation. Indicated.

% Inhibition = 100 [1-CPM (T) -CPM (C2) / CPM (C1) -CPM (B)]

(CPM: count per minute)

CPM (T): CPM when sample and enzyme are added

CPM (1): CPM when no sample is added and enzyme is added

CPM (C2): CPM when sample is added but enzyme is not

CPM (B): CPM without sample and enzyme

compound ACAT inhibitory activity (%) One 22.2 2 11.7 3 46.4

As shown in Table 4, the ACAT inhibitory activity of Compound 1, Compound 2, and Compound 3 was 22.2%, 11.7%, and 46.4%, respectively, indicating that ACAT inhibitory activity was present. That is, it was confirmed that Compound 1, Compound 2 and Compound 3 isolated in the present invention can be used as a cardiovascular disease treatment agent.

3-2) Determination of DGAT Enzyme Inhibitory Activity of Sesquiterpene Compounds

As an additional experiment to determine whether the sesquiterpene lactone compound isolated and purified from the white mudfish in Example 1 can be used for cardiovascular disease treatment, the DGAT enzyme inhibitory activity of each sesquiterpene lactone compound was measured.

DGAT activity was measured by using a rat liver microsome as an enzyme and ¹⁴ C-ole into TG (triacylglycerol) with 1,2-dioleoyl-sn-glycerol added externally. It was determined by measuring the influx of oil-CoA (oleoyl-CoA). The reaction solution (final volume; 200 μl) was 100 mM Tris-HCl, 1 mM DTT, 50 mM NaF, 5 μM essentially fatty acid free (BSA), 300 μM 1,2-dioleoyl-sn-glycerol, 40 μM ¹⁴ C-oleoyl CoA (0.1 μCi / tube) and 10 μl of a compound sample dissolved in DMSO, and the enzyme reaction was performed at 37 ° C. for 10 minutes. After stopping the reaction by adding 1.25 ml of isopropanol: heptane (1: 1) mixed solution, 600 µl of organic phase was obtained, transferred to a new tube, and dried completely using a speed vacuum concentrator. . After separating only the fat part from the organic layer using TLC, only the neutral fat band part was scraped off and the amount of the neutral fat was measured using a liquid scintillation counter. By the above measurement, the activity of DGAT could be measured. DGAT enzyme inhibitory activity of each compound was expressed as a percentage of the enzyme activity in the sample containing only DMSO.

As a result, in the case of compound 1 and compound 2, DGAT inhibitory activity was found to be 38.6% and 51.1%, respectively, indicating that there was DGAT activity.

<Example 4>

Determination of FPTase Inhibitory Activity of Sesquiterpene Compounds

FPTase enzyme inhibitory activity of each sesquiterpene lactone compound was measured in order to determine whether the sesquiterpene lactone compound isolated and purified from white bark can be used for cancer treatment.

FPTase activity was measured using SPA kit (Scintillation Proximity Assay kit, Amersham Pharmacia Biotech). FPTase activity measures the transfer of [ ³ H] -panesyl groups from [ ³ H] -farnesyl pyrophosphate to biotin-KKKSKTKCVIM (Ki-ras c-terminal sequence as substrate of FPTase). Is determined by. The reaction solution (final volume; 100 μl) was 50 mM HEPES (pH 7.5), 30 mM MgCl ₂ , 20 mM KCl, 5 mM DTT, 0.01% Triton X-100, 150-250 nM [ ³ H] -Panesyl pyrophosphate (60 μM, 1 Ci / μl), 25-50 ng of recombinant rat FPTase, 10-200 nM Biotin-KKKSKTKCVIM, 10 μl of a compound sample solution (1-100 mg / 1 ml DMSO) dissolved in DMSO. After reacting at 37 ° C. for 60 minutes, the reaction was stopped by addition of 150 μl of stop / bead solution. The mixture was mixed well again and allowed to stand for 30 minutes at room temperature, and FPTase activity was measured by measuring the amount of [ ³ H] -panesyl transferred to biotin-KKKSKTKCVIM using a radiometer (1450 Microbeta counter, Wallac). Measured. Enzyme inhibitory activity was calculated as% of the enzyme activity in the sample containing only DMSO, the results are shown in Table 5 below.

compound % Of FPTase inhibitory activity One 69.7 2 58.0 3 29.6

As shown in Table 5, the FPTase inhibitory activity of Compound 1, Compound 2 and Compound 3 was found to be 69.7%, 58.0% and 29.6%, respectively, indicating that there was FPTase inhibitory activity. That is, it was confirmed that Compound 1, Compound 2, and Compound 3 can be used as cancer therapeutic agents.

<Example 5>

Preparation of Pharmaceutical Formulations Comprising Compounds of the Invention

The following formulation examples are merely illustrative of the formulation of the present invention and do not limit the scope of the invention. Hard gelatin capsules were prepared using the ingredients in Table 6 below.

ingredient Content (mg / capsule) Active Ingredients (Compounds of the Invention) 50 Vitamin c 50 Carriers or excipients (starch, lactose, etc.) 380 Magnesium stearate 20 Total amount 500

The ingredients were mixed thoroughly to fill the hard capsules in a total amount of 500 mg.

The composition may further include other health-neutral ingredients in addition to vitamin C, and various oral preparations, such as tablets, granules, powders, microcapsules, and drinks, according to methods generally known using the composition. And parenteral preparations such as various injections.

As described above, the selusquiterpene lactone compound extracted from white bark, zaluzanin C, 9α-hydroxyguaian-4 (15), 10 (14), 11 (13) -triene-6,12- Olid and Eryrin M have ACAT inhibitory activity, DGAT inhibitory activity and FPTase inhibitory activity. Therefore, the compound can be used as a composition for the prevention and treatment of cardiovascular diseases and cancer.

Claims (7)

Zaluzanin C of formula 1, 9α-hydroxyguaian-4 (15), 10 (14), 11 (13) -triene-6,12-oxide of formula 2 containing a sesquiterpene lactone compound selected from the group consisting of hydroxyguaian-4 (15), 10 (14), 11 (13) -triene-6,12-olide) and ixerin M of Formula 3 , Composition for preventing and treating cardiovascular diseases due to hypercholesterolemia or hypertriglyceridemia. <Formula 1>

<Formula 2>

<Formula 3>

According to claim 1, wherein the cardiovascular disease is a composition for preventing and treating cardiovascular disease, characterized in that selected from the group consisting of arteriosclerosis, hypertension, angina pectoris, hyperlipidemia, myocardial infarction and heart failure. The method of claim 1, wherein the zaluzanin C, 9α-hydroxyguaian-4 (15), 10 (14), 11 (13) -triene-6,12-oxide (9α-hydroxyguaian -4 (15), 10 (14), 11 (13) -triene-6,12-olide) and ixerin M extract the root diameter of Ixeris dentata forma albiflora with C1-C5 alcohol. Cardiovascular disease prevention and treatment composition, characterized in that obtained by. The method of claim 3, wherein after extracting with C1 ~ C5 alcohol, the extract is fractionated with ethyl acetate, water, butanol, and then further comprising silica gel column chromatography to perform a cardiovascular disease prevention and treatment composition. delete delete delete

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