KR20030053178A - AN ACYL CoA CHOLESTEROL ACYLTRANSFERASE INHIBITORS AND A THERAPEUTIC AGENT CONTAINING PHEOPHORBIDE A OF PORPHYRIN COMPOUND OR AN EXTRACT OF PERSICARIA VULGARIS AS AN EFFECTIVE INGREDIENT FOR THE TREATMENT OF HEART DISEASE - Google Patents

Abstract

PURPOSE: A compound containing pheophorbide a as a porphyrin compound and a Persicaria vulgaris extract as an active ingredient is provided. The pheophorbide a inhibits synthesis of very low density lipoprotein in liver and lowers blood cholesterol level and thus can be effectively used as a prophylactic and therapeutic agents for prevention and treatment of cardiovascular disease such as hyperlipidemia, arteriosclerosis caused by hypercholesterolemia. CONSTITUTION: An inhibitor for activity of acyl-coa:cholesterol acyltransferase(ACAT) contains porphyrin-based pheophorbide a represented by the formula(1). The inhibitor for activity of ACAT contains a Persicaria vulgaris extract containing the porphyrin-based pheophorbide a. The Persicaria vulgaris extract is prepared by extracting in C1-4 alcohol and reextracting in an organic solvent or water. The organic solvent is selected from ethyl acetate, N-hexane or chloroform. The porphyrin-based pheophorbide a or Persicaria vulgaris extract has an LD50 of 3.2 g/kg.

Description

Activation inhibitor of acyl coeicholesteryl acyltransferase containing porphine-based compound or extract of spring-like extract as an active ingredient and preventive and therapeutic agent for cardiovascular disease PORPHYRIN COMPOUND OR AN EXTRACT OF PERSICARIA VULGARIS AS AN EFFECTIVE INGREDIENT FOR THE TREATMENT OF HEART DISEASE}

The present invention relates to the use of a porphyrin-based compound represented by the following formula (1), which is a porphyrin-based compound (pheophorbide a) and the spring extract, more specifically, the spring and the porphine-based compound Fairpovid A. Extract specifically inhibits the activity of Acyl-CoA: Cholesterol acyltransferase (hereinafter referred to as “ACAT”), thereby preventing cardiovascular diseases such as hyperlipidemia and arteriosclerosis caused by hypercholesterolemia. It can be used for prevention and treatment.

Formula 1

Cholesterol required by living organisms include exogenous cholesterol due to food intake and endogenous cholesterol obtained by synthesizing cholesterol from liver in vivo [Heider JG JR Prous Science , 1986, 423-438].

Too much cholesterol in the body causes abnormalities in the formation, transport, and degradation of lipoproteins, and abnormal metabolism of the lipoproteins leads to excessively high blood cholesterol, leading to atherosclerosis.

Recently, the rate of death from cardiovascular disease caused by hypercholesterolemia accounts for the highest rate among all mortality rates. As a result of epidemiologic investigations, ischemic heart disease is caused by elevated atherosclerosis of the coronary artery. Sclerosis is mainstream.

To lower blood cholesterol, methods have been proposed to inhibit cholesterol absorption in the small intestine, to inhibit the biosynthesis of cholesterol in the liver itself, or to promote the excretion of bile acids. [Goldstein JL and SM Brown Nature , 1990, 33 , 425-430 , Komai T. and Y. Tsujita DN & P , 1994, 7 , 279-288].

In practice, most medicines lower the blood cholesterol level by inhibiting the biosynthesis of cholesterol in the liver as described above. For example, the biologics of the compactin marketed by Sankyo, Japan, and Merck, USA Modified derivatives, pravastatin and simvastatin, have the highest market share and elongation.

In addition, the concentration of cholesterol in the blood by inhibiting the reductase of 3-hydroxy-3-methyl glutaryl Co-A (hereinafter referred to as "HMG Co-A"), which is involved in the intermediate stage of the synthesis of cholesterol in the liver. Lowering method is also used.

However, if the HMG Co-A reductase inhibitor is used for a long time, ubiquinone, dolichol, haem A, which are necessary for the human body generated in the intermediate stage of cholesterol synthesis after mevalonate, ), It has been reported to affect the production of steroid hormones, vitamin D, bile acids and lipoproteins produced from farnesylated protein and cholesterol [Grunler J., J. Ericsson and G. Dalloner Biochim. Biophys, Acta 1994, 1212 , 259-277].

Indeed, Willis found that continued use of these HMG Co-A reductase inhibitors reduced the synthesis of coenzyme cues, which play an important role in cardiac and immune function, which could adversely affect arteriosclerosis patients and heart disease patients. Reported [Willis RA, K., Folkers. JL Tucker, CQ Ye, LJ Xia, and H Tamagawa Proc. Natl. Acad. Sci ., 1990, 87 , 8928-8930].

In addition, bile acid, a high concentration of cholesterol, is secreted from the liver to digest food and reabsorbed in the large intestine.The method of binding anion exchanger to the secreted bile acid and inhibiting resorption of cholesterol is clinically used as a therapeutic agent for It is used.

As described above, the development of a new cardiovascular treatment agent with a certain mechanism of action, less side effects, and no restrictions on use has been required, and ACAT activity inhibitors have attracted attention as a preventive and therapeutic agent for cardiovascular diseases caused by hypercholesterolemia [Sliskovic]. DR and AD White Trends in Pharmacol. Sci ., 1991, 12 , 194-199]

The ACAT enzyme is an enzyme that inhibits the absorption of cholesterol in the small intestine by inhibiting the acyl metabolism of cholesterol and inhibits the synthesis of very low density lipoprotein (VLDL) in the liver. In addition, the enzyme inhibits the progression of atherosclerosis by inhibiting the accumulation of storage-type cholesterol in adipocytes and blood vessel lining.

In fact, foreign research institutes, universities, and pharmaceutical companies are developing ACAT activity inhibitors as a next-generation drug that directly inhibits the mechanism of atherosclerosis as a prophylactic and therapeutic agent for cardiovascular disease caused by hypercholesterolemia.

Among them, there are some drug candidates in the pre-clinical stage after the in vivo activity test, but there is no ACAT activity inhibitor currently used in the clinic.

Most of the ACAT inhibitors studied so far are chemically synthesized compounds, for example, urea compounds and amide compounds are predominant [Matsuda K. 1994. ACAT inhibitors as antiatherosclerosis agent: compounds and mechanisms.14, John Wiley & Son, Inc., 271-305.

Recently, in the development of medicines, as part of clinical trials to reduce side effects and develop leading substances, exploration is being conducted on microbial resources. An example of the research is as follows. Of purpactin (Tomoda H., H. Nishida, R. Masuma, J. Cao, S. Okuda and S. Omura J. Antibiotics , 1991, 44 , 136-143) at Kitasato Institute, Japan Beginning with the revealed structure, epi-cohliquinone A (Japanese Patent Laid-Open No. 4-334383) by Sankyo, Japan, and acatelin [Naganuma S., K Sakai, K] Hasumi and A. Endo J. Antibiotics , 1992, 45 , 1216-1221], helmintosporol [Park JK, K. Hasumi and A Endo J. Antibiotics , 1993, 46 , 1303-1305], d. Territin (Hasumi K., C. Shinohara, T. Iwanaga and A. Endo J. Antibiotics , 1993, 46 , 1782-1787), gypsetin [Shinohara C., K. Hasumi, Y. Takei and A. Endo J. Antibiotics , 1994, 47 , 163-167], enniatins of the Kitasato Institute of Japan [Nishida H., XH Huang, R. Masuma, YK Kim and S. Omura J. Antibiotics , 1992, 45 , 1207-1214], glisoprenins [Tomoda, HX H, Huang, H. Nishida, R Masuma, YK Kim and S. Omura J. Antibiotics , 1992., 45, 1202-1206], pyripyropenes [Omura S., H. Tomoda, YK Kim and H. Nishida J. Antibiotics , 1993, 46 , 1168-1169 ; Kim Y. K, H Tomoda, H. Nishida, T. Sunazuka, R. Obata, S. Omura J. Antibiotics , 1994, 47 , 154-162], terpendols [Huang X. H, H. Tomoda , H. Nishida, R, Masuma and S. Omura J. Antibiotics , 1995, 48 , 1-4], AS-183 by Kyowa Hakko, Japan [Kuroda K., M. Yoshida, Y. Uosaki, K. Ando, I. Kawamoto, E. Oishi, H. Onuma, K. Yamada and Y. Matsuda J. Antibiotics , 1993, 46, 196-1202], AS-186 [Kuroda K., Y. Morishita, Y. Saito, Y. Ikuina, K. Ando, I. Kawamoto and Y. Matsuda J. Antibiotics , 1994, 47 , 16-22], GERI-BP-001 of the Korea Research Institute of Bioscience and Biotechnology [TS Jeong, SU Kim, K. H. Son, BM Kwon, YK Kim, MU Choi and SH Bok J. Antibiotics , 1995, 48 , 751-756], GERI-BP-002 [YK Kim, HW Lee, KH Son, BM Kwon, TS Jeong, DH Lee, JH Shin, YW Seo, SU Kim, SH Bok J. Antibiotics , 1996, 49 , 31-36].

Accordingly, the present inventors, while trying to obtain a new leading material having an ACAT activity inhibitory effect, isolating and purifying the active material from the natural resources of springtime to obtain the porphine compound of the formula (1) of the fair forbidden A, the compound The present invention was completed by revealing this concentration-dependent inhibition of ACAT activity.

It is an object of the present invention to provide a use of an inhibitor of ACAT activity containing a porphine-based compound of Formula 1 as an active ingredient, an extract containing evergreen pair and a spring extract containing the same.

It is also an object of the present invention to provide a preventive and therapeutic agent for cardiovascular diseases caused by hypercholesterolemia, which comprises a porphine-based compound of Formula 1, fairpovid A and a spring extract containing the same as an active ingredient.

1 is a structure of a fairovide A, a porphine-based compound represented by Formula 1 of the present invention,

Figure 2 shows the hydrogen-nuclear magnetic resonance spectrum (CDCl ₃ , 500.13 MHz) of the porphinide A, a porphine-based compound represented by the formula (1) of the present invention,

Figure 3 shows the carbon-nuclear magnetic resonance spectrum (CDCl ₃ , 125.75 MHz) of the porphine-based compound represented by Formula 1 of the present invention,

Figure 4 shows the DEPT spectrum (CDCl ₃ , 125.75 MHz) of the porphinide A, a porphine-based compound represented by Formula 1 according to the present invention,

FIG. 5 is a diagram showing an HMBC-related picture of the porphovide A, a porphine-based compound represented by Chemical Formula 1 of the present invention.

Figure 6 shows the ACAT inhibitory activity of the fair forbidden A, a porphine-based compound represented by the formula (1) of the present invention.

In order to achieve the above object, the present invention provides a use of an inhibitor of ACAT containing a porphine-based compound represented by the following formula (1) and a spring extract containing the same as an active ingredient.

Formula 1

A porphine-based compound comprising Formula 1 can be purified and separated in spring ( Persicaria vulgaris ), the spring is extracted with alcohol, the extract obtained by re-extracting with an organic solvent, the ash obtained Obtained by separating and purifying the extract.

The spring extract extracted with alcohol in the spring is possible by immersion, cold immersion, or the method of warming, but is preferably cold immersion at room temperature for 3 days.

The alcohol used is selected from C ₁ to C ₄ alcohols and used, preferably methanol is used.

In addition, the organic solvent to be re-extracted in step 2 is selected from ethyl acetate, normal- hexane and chloroform and used, preferably ethyl acetate.

The extract obtained in the above step can be separated and purified using a method commonly used by those skilled in the art, and the present invention is carried out by repeating chromatography two to three times, preferably Preferably, the chromatography is separated and purified using Sephadex L-20.

Figure 1 shows the structure of the compound finally separated and purified by the method of the present invention, as measured by UV-visible spectrophotometer, peaks of 408 nm and 666 nm observed in the visible region of the characteristic peak of the porphine-based compound By showing a dark green color, it is possible to confirm the presence of the porphine ring in the structure of the compound. In addition, the result of measuring the molecular weight ([M + H] ⁺ ) was m / z 593.2767, which showed an error of 0.3 mD with the theoretical calculation. As a result of the measurement of the high resolution FAB-MS, the molecular formula was C ₃₅ H ₃₆ O. Expected to be ₅ N ₄ .

In order to determine the structure of the isolated compound, Figure 2 is a hydrogen-nuclear magnetic resonance spectrum (CDCl ₃ ) and Figure 3 using a nuclear magnetic resonance group of 500.13 MHz for the porpoid A, a porphine-based compound represented by Formula 1 Is the carbon-nuclear magnetic resonance spectrum (CDCl ₃ ) using a nuclear magnetic resonance at 125.75 MHz.

As shown in the results of FIG. 2, δ9.29 is the lowest magnetic field of the vinyl group methine proton of the unsaturated chlorin ring of the C ring most characteristic of the porphine-based compound structure of the present invention , Observed in 9.13 and 8.43.

In addition, the methyl protone of the methyl group respectively bonded to C-4, C-8, C-13, and C-18 represents δ1.72, 3.23, 3.00 and 3.53, respectively, and is bound to C-14. Hydrogen of the ethyl group (ethyl proton) is shown at δ 3.44 and 1.52, and hydrogen of another olefin group linked to 28-H at δ6.01 is 28-H, the hydrogen of one olefin group (olefinic proton) at the end ( 27-H, an olefinic proton, was found at δ7.77.

The hydrogen peak of -NH inside the structure, which is another feature of the porphine structure, was observed at δ-1.92, indicating that hydrogen inside the porphine was strongly blocked in the magnetic field region.

FIG. 4 is a diagram illustrating a distortionless enhancement by polarization transfer (DEPT) spectrum (CDCl ₃ , 125.75 MHz), which is not distorted by the polarization shift of the porphinide A, a porphine-based compound represented by Chemical Formula 1, wherein 6 methyl, Three methylenes, three methines, one sp ² methylene, four sp ² methines, 15 sp ² quaternary carbons, and three carbonyl carbons were observed.

Figure 5 depicts the ^{HMBC (1 H- 13 C-heteronuclear} multiple-bond correlation spectroscopy) of the pair Poby de this formate pingye compound represented by the general formula (I) of the present invention, δ2.54 (2H, m, 1 -H ) Correlation from methylene to δ178.05 (s, C-35), 51.01 (d, C-3) and 29.54 (t, C-2) carbon, from 2.15 (2H, m, 2-H) methylene. Correlation to 05 (s, C-35), 161.06 (s, C-23), 51.01 (d, C-3), 50.07 (d, C-4) and 30.82 (t, C-1) carbon, 4.11 (1H, m, H-3) from methine 172.03 (s, C-5), 161.06 (s, C-23), 50.07 (d, C-4), 30.82 (t, C-1), 29.54 (t , C-2) and correlation to 23.09 (q, C-25) carbon, 172.03 (s, C-5), 161.06 (s from 4.34 (1H, dq, J = 7.2, 1.9 Hz, H-4) methine , C-23), 51.01 (d, C-3), 29.54 (t, C-2) and 23.09 (q, C-25) carbon to carbon, 1.72 (3H, d, J = 7.2 Hz, H- 25) Correlation from methyl to 172.03 (s, C-5), 51.01 (d, C-3) and 50.07 (d, C-4) carbon, 8.43 (1H, s, H-6) 172.03 (s from methine , C-5), 142.00 (s, C-7), 131.79 (s, C-8) and the correlation to 50.07 (d, C-4) carbon, 142.00 (s, C-7), 136.06 (s, C-9) and 131.79 (3.23 (3H, s, H-26) methyl) s, C-8) Correlation to carbon, 7.77 (1H, dd, J = 17.8, 11.5 Hz, H-27) methine from 136.15 (s, C-10), 136.06 (s, C-9), 131.79 ( s, C-8) and correlation to 122.65 (t, C-28) carbon, 6.01 (1H, dd, J = 17.8, 1.2 Hz, Ha-28), 6.11 (1H, dd, J = 11.5, 1.2 Hz , Hb-28) correlation from methylene to 136.06 (s, C-9) and 128.95 (d, C-27) carbons, 9.13 (1H, s, H-11) methine from 155.56 (s, C-12), 136.43 (s, C-13), 136.15 (s, C-10) and 136.06 (s, C-9), correlation to carbon, 3.00 (3H, s, H-29) from methyl 155.56 (s, C- 12), 145.09 (s, C-14) and 136.43 (s, C-13) correlation to carbon, 3.44 (2H, q, J = 7.3 Hz, H-30) 150.88 (s, C-15) from methylene , 145.09 (s, C-14), 136.43 (s, C-13) and 17.33 (q, C-31) correlation to carbon, 1.52 (3H, d, J = 7.2 Hz, H-31) methyl from 145.09 Correlation to (s, C-14) and 19.32 (t, C-30) carbons, 9.29 (1H, s, H-16) me From 150.88 (s, C-15), 145.09 (s, C-14), 137.86 (s, C-17) and 128.95 (s, C-18) to carbon, 3.53 (3H, s, H-32 ) Correlation from methyl to 137.86 (s, C-17) and 128.95 (s, C-19) carbons, 6.14 (1H, s, H-21) methine to 189.56 (s, C-20), 169.60 (s, C-33), 161.06 (s, C-23), 149.61 (s, C-24), 128.95 (s, C-19) and 105.11 (s, C-22) correlation to carbon and 3.78 (3H, s , H-34) correlation from methyl to 169.60 (s, C-33) carbon was observed.

From the above analysis results, it can be expected that the compounds extracted and separated and purified in the spring of the present invention are porphine-based compounds having a chlorin macrocycle structure. As a result of observing with the same spectrophotometer method, it was confirmed that the compound obtained in the present invention was a pheophorbide a of the porphine-based compound represented by the formula (1).

Figure 6 shows the results of measuring the ACAT inhibitory activity of the porphin-A compound of the formula (1), IC ₅₀ (inhibition concentration) value is 2.2 μM, the inhibitory activity increases as the concentration of the fair forbidden A increases By showing a proportional relationship, it can be used as an agent for preventing and treating cardiovascular disease caused by hypercholesterolemia.

In addition, the present invention provides an agent for preventing and treating cardiovascular diseases caused by hypercholesterolemia, which comprises a porphine-based compound of Formula 1, fairpovid A, and a spring extract containing the same as an active ingredient.

The porphinide compound of the present invention, or fair spring bead containing the same extract may be administered in an effective amount through various routes of administration. The use together contains a pharmaceutically acceptable carrier. More specifically, pharmaceutically acceptable carriers can be any standard pharmaceutical carrier that can be used in known formulations such as sterile solutions, tablets, coated tablets and capsules. Typically, the carrier may include excipients such as starch, milk, sugar, certain types of clays, gelatin, stearic acid, talc, vegetable oils or oils, gums, glycols, or other known excipients, and also flavors, color additives And other ingredients.

The formulation for administering the composition containing the porphine A or spring extract of the present invention as an active ingredient as an active ingredient in the above range can be oral, intravenous, intramuscular, or transdermal administration in a conventional manner. And may be provided in the form of general pharmaceutical formulations, but are not limited to these methods.

The porphinide compound of the present invention can be administered in a variety of oral and parenteral formulations in actual clinical administration, when formulated, fillers, extenders, binders, wetting agents, disintegrants, surfactants that are commonly used It is prepared using diluents or excipients. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient such as porphin-based compound Fairpovid A or spring extract, for example starch, calcium carbonate. It is prepared by mixing (calcium carbonate), sucrose or sucrose, lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium styrene 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 oral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilizers, suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters 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, the pharmaceutical compositions of the present invention can be administered parenterally, and parenteral administration is by subcutaneous injection, intravenous injection or intramuscular injection. In order to formulate into a parenteral dosage form, the porphine-based compound, fairpovid A or spring extract, is mixed in water with a stabilizer or buffer to prepare a solution or suspension, which is prepared in a unit dosage form of ampoules or vials. do.

The dosage of the active ingredient according to the present invention is appropriately selected according to the absorbance of the active ingredient in the body, the rate of water activation and excretion, the age, sex and condition of the patient, the severity of the disease to be treated, but generally 1 per day It can be administered several times or several times, and in the case of the porphin-based compound Fair Forbidden A is 10 to 50 mg / kg, preferably 15 to 20 mg / kg, spring to extract 100 ~ 500 mg / kg And preferably 150 to 200 mg / kg. The exact amount, route of administration, and frequency of the agent can be readily determined according to the nature of the agent, the weight and condition of the subject to be administered, and the nature of the particular derivative to be used.

The porphine A or spring extract of the present invention did not show toxicological changes up to 0.1 g / kg in experimental mice, and the minimum lethal dose (LD ₅₀ ) of 1.0 g / kg or higher was high in bioavailability. Can be safely administered.

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

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

Example 1 Isolation and Purification of Porphine Compounds from Spring Extracts

Spring was gathered in the valley of Bomunsan near Daejeon, and the collected spring outpost was verified by a plant classification expert, dried in the shade, and chopped. The dried spring was cooled to 3 times methanol by weight at room temperature for 3 days and then filtered. The filtrate was concentrated under reduced pressure to obtain an extract. The spring extract was prepared using the normal-hexane, chloroform, ethyl acetate and water each fraction to measure the ACAT inhibitory activity by the method of Experimental Example 1 below.

As a result of measuring the ACAT inhibitory activity by the method of Experimental Example 1, the normal-hexane fraction (100 ㎍ / ㎖) 40%, chloroform fraction (100 ㎍ / ㎖) 20%, ethyl acetate fraction (100 ㎍ / ㎖) ) Exhibited ACAT inhibitory activity of 60% and water fraction (100 μg / ml).

Among them, ethyl acetate fraction having the highest ACAT inhibitory activity was selected and concentrated under reduced pressure, and column chromatography of silica gel was performed using a step gradient solvent system composed of chloroform: methanol (100/0 to 0/100). (Column Chromatography) and the fraction obtained by measuring the ACAT inhibitory activity, and the fractions with a high inhibitory activity of 75% or more at a concentration of 50 ㎍ / ㎖, using the elution solvent of CHCl ₃ : MeOH = 1: 1 Sephadex LH-20 chromatography was performed. The resulting fractions were subjected to HPLC (Column: YMC-pack SIL, 250 × 20 mm), flowing chloroform: methanol = 97: 3 at 5 ml / min as the elution solvent, and each fraction obtained was 254 nm on an ultraviolet spectrometer. Fixed to search for the active substance. At this time, it was confirmed that the part eluted at 19.9 minutes is a pure compound showing the enzyme inhibitory activity.

Dark green powder; UV _max : 408 nm and 666 nm; FAB-MS (+) Mass (m / z): 593 [M + H] ⁺ ; HRFAB-MS (m / z) 593.2767; ¹ H NMR (CDCl ₃ ): δ 1.52 (3H, d, J = 7.2 Hz, H-31), 1.72 (3H, d, J = 7.2 Hz, H-25), 2.15 (2H, m, 2-H ), 2.54 (2H, m, 1-H), 3.00 (3H, s, H-29), 3.23 (3H, s, H-26), 3.44 (2H, q, J = 7.3 Hz, H-30) , 3.53 (3H, s, H-32), 3.78 (3H, s, H-34), 4.11 (1H, m, H-3), 4.34 (1H, dq, J = 7.2, 1.9 Hz, H-4 ), 6.01 (1H, dd, J = 17.8, 1.2 Hz, Ha-28), 6.14 (1H, dd, J = 11.5, 1.2 Hz, Hb-28), 6.14 (1H, s, H-21), 7.77 (1H, dd, J = 17.8, 11.5 Hz, H-27), 8.43 (1H, s, H-6), 9.13 (1H, s, H-11), 9.29 (1H, s, H-16), -1.92 (br, s, NH) and ¹³ C-NMR: δ 189.56 (s, C-20), 178.05 (s, C-35), 172.03 (s, C-5), 169.60 (s, C-33 ), 161.06 (s, C-23), 155.56 (s, C-12), 150.88 (s, C-15), 149.61 (s, C-24), 145.09 (s, C-14), 142.00 (s , C-7), 137.86 (s, C-17), 136.43 (s, C-13), 136.15 (s, C-10), 136.06 (s, C-9), 131.79 (s, C-8) , 128.95 (s, C-18), 128.95 (s, C-19), 128.95 (d, C-27), 122.65 (t, C-28), 105.11 (s, C-22), 104.31 (d, C-16), 97.43 (d, C-11), 93.06 (d, C-6), 64.64 (d, C-21), 52.85 (q, C-34), 51.01 (d, C-3), 50.07 (d, C-4), 30.82 (t, C-1), 29.54 (t, C-2), 23.09 (q, C-25), 19.32 (t, C-30), 17.33 (q, C-31), 12.05 (q, C-32), 12.01 (q, C-26), 11.10 (q, C-29).

Preparation Example 1 Preparation of ACAT Enzyme Source

ACAT Liver from rats (Male Sprague-Dawley rats 250-300 g) was enzymatically washed with microsomal buffer A (0.25 M sucrose, 1 mM EDTA, 0.01 M tris-hydrochloric acid, pH 7.4) and scissors. 1-2 cm³It was sliced to size and homogenized using a homogenizer consisting of Teflon-glass. The homogenate was centrifuged at 14,000 × g for 15 minutes to collect the supernatant and again centrifuged at 100,000 × g for 1 hour. To isolate microsomes containing ACAT enzyme activity, the centrifuged precipitate was added to microsome buffer B (0.25 M sucrose, 0.01 M tris-hydrochloric acid, pH 7.4) for 1 hour at 100,000 × g. Centrifugation again. In order to homogenize the protein concentration of the enzyme source in the test, the microsomal buffer B is appropriately dissolved in the centrifuged precipitate, and the protein is prepared by the Lowry method using bovine serum albumin as the protein standard. After the concentration was determined, the enzyme source was diluted with microsomal buffer B, the protein concentration was adjusted to 10 mg / ml, and dispensed into 1 ml vials and stored at -70 ° C.

Experimental Example 1 ACAT Enzyme Activity Measurement

The ACAT enzyme inhibitory activity of the porphine-paired forbidden A of the present invention was examined.

ACAT enzyme activity was determined by using [ ^1-14 C] oleoyl-coei as a substrate and some modifications to the following known methods [YKKim, HW Lee, K. H Son, B. M Kwon, TS Jeong, DH Lee, JH Shin, YW Seo, SU Kim, SH Bok, J. Antibiotics , 1996, 49 , 31-36].

As the reaction solution, 10.0 μl of sample solution, 4.0 μl of liver microsomal enzyme from liver tissue, standard buffer [0.5 M KH ₂ PO ₄ , 10 mM DTT (Dithiothreitol), pH 7.4] 20.0 μl, bovine serum albumin was used as an essentially fatty acid free grade to prevent excess cholesterol from entering the screening system. 15.0 μl of 40 mg / ml, 2.0 μl of 20 mg / ml cholesterol, distilled water 41.0 μl was added and preliminarily reacted at 37 ° C. for 20 minutes. 8.0 μl of [ ^1-14 C] oleoyl-coei (0.05 μCi, final concentration of 10 μM) was added to the reaction solution, followed by reaction at 37 ° C. for 25 minutes, followed by 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 5-fold standard buffer were added and centrifuged.

ACAT enzyme activity was measured by adding 3 ml of scintillation cocktail to 100 μl of the supernatant obtained by centrifugation, followed by measuring the radioactivity using a liquid scintillation counter.

The ACAT enzyme inactivation activity was measured by using a radiometric measuring device by placing a search sample on a substrate and an enzyme labeled with radioactivity, and calculating the ACAT enzyme inactivation activity using Equation 1 below.

% Deactivation = 100 × [1-{CPM (T) -CPM (C2) / CPM (C1) -CPM (B)}]

CPM (T): CPM (counter per minute) when sample and enzyme are added

CPM (C1): No sample added, enzyme added CPM

CPM (C2): CPM when sample was added but no enzyme was added

CPM (B): CPM without sample and enzyme

At this time, the reaction was carried out at 0 ° C., and obovatol was used as a positive control.

As a result of measuring the ACAT enzyme inhibitory activity using Equation 1, the IC ₅₀ value of the obovatol, which is a positive control, is 44 μM, and the Fairfin bead of the porphine-based compound represented by Formula 1 of the present invention. The IC ₅₀ value was 2.2 μM and also showed ACAT enzyme inhibitory activity depending on the concentration of Fairfur A used (see FIG. 6).

ACAT Enzyme Inhibitory Activity of Pairfervid A Concentration (µg / ml) ACAT inhibitory activity (%) SD 100 91.9 ± 0.88 33 84.7 ± 1.65 11 79.2 ± 0.59 3 67.2 ± 1.83 One 48.2 ± 1.98 0.4 36.1 ± 0.11 0.1 33.2 ± 6.51

From the above results, as a result of measuring the ACAT enzyme inactivation activity against the porphine-fairovide A of Formula 1 of the present invention, the inhibitory activity was shown to be concentration-dependent, and the IC ₅₀ value was 2.2 μM, which is stronger than the positive control. Inhibitory effect was confirmed. It has been shown to inhibit cholesterol absorption in the blood vessels by inhibiting cholesterol metabolism metabolism in blood vessels, thereby lowering the cholesterol concentration in the blood, and because it is obtained from natural resources rather than synthesized compounds, it has been shown to be a conventional cholesterol synthesis inhibitor. May alleviate side effects. Therefore, it can be usefully used as a prophylactic and therapeutic agent for cardiovascular diseases such as hyperlipidemia and atherosclerosis, which is caused by hypercholesterolemia, which includes porphyrin-based fairpoid A of Formula 1 and spring extracts including the same as an active ingredient. have.

Experimental Example 2 Oral Acute Toxicity Test in Experimental Mice

Acute toxicity was investigated in the following manner to clinically utilize the fair forbidden A of the formula (1).

The 6-week-old SPF rats were divided into 5 rats per group, and the Fairpoid A of Formula 1 of the present invention was suspended in 0.5% methylcellulose solution and administered orally at a dose of 0.1 g / kg. It was. After administration, the mortality, clinical symptoms, and weight changes of the animals were observed. Hematological and hematological and biochemical tests were performed.

As a result, there were no clinical symptoms or dead animals in the animals treated with the Fairpoid A of Formula 1, and no toxicity change was observed in weight change, blood test, blood biochemical test, autopsy findings, etc. . The compound tested did not show toxic changes up to 0.1 g / kg in rats, and the minimum lethal dose (LD ₅₀ ) was determined to be a safe substance of 0.1 g / kg or more.

Preparation Example 1 Preparation of Tablet

Tablets containing 1 g of Fairpoid A of the present invention as an active ingredient are prepared by the following method.

1.00 g of Fairpovid A was mixed with 7 g of lactose, 1.5 g of crystalline cellulose, and 0.5 g of magnesium stearate, and then a tablet was prepared by a direct tableting method. The total amount of each tablet is 100 mg, of which the active ingredient content is 10 mg.

The components of the tablet are as follows.

1 g of Fair Forbidden A ...

Lactose ... 7 g

Crystalline cellulose 1.5 g

Magnesium Stearate0.5 g

Total amount: 10 g

Preparation Example 2 Preparation of Capsule

Capsules containing 1 g of Fair Forbidden A of the present invention as an active ingredient are prepared by the following method.

A powder was prepared by mixing 1.00 g of Fairford Bead A with 5.00 g of corn starch and 10.0 g of carboxy cellulose. 100 mg of the powder was put in a hard capsule to prepare a capsule.

The components of the capsules are as follows.

1 g of Fair Forbidden A ...

5 g of corn starch ··············

Carboxy cellulose 4 g

Total amount: 10 g

As discussed above, porphine-paired forbidden A of the present invention inhibits ACAT enzyme activity, unlike conventional drugs developed by inhibiting cholesterol biosynthesis itself, by inhibiting cholesterol acyl transfer metabolism. It is effective in lowering cholesterol levels in the small intestine and inhibits the synthesis of very low density lipoprotein (VLDL) in the liver, thereby lowering low density lipoprotein (LDL) cholesterol and blood cholesterol levels. Can be lowered. In addition, as a compound obtained from a natural plant, conventionally used cholesterol synthesis inhibitors can alleviate side effects caused by the synthetic compound, effective for the porphin-based compound of Formula 1 of the present invention and the spring extract containing the same It can be usefully used as an agent for preventing and treating cardiovascular diseases such as hyperlipidemia and arteriosclerosis caused by hypercholesterolemia.

Claims (6)

Inhibitor of the activity of acyl-CoA: cholesterol acyltransferase (ACAT) containing porphine-based fairovidide a represented by the following formula (1) as an active ingredient Formula 1 A prophylactic or therapeutic agent for cardiovascular disease caused by hypercholesterolemia, comprising the porphine-type fairpoid A of claim 1 as an active ingredient. Spring extract ( Persicaria vulgaris ) is extracted with alcohol, and the extract is extracted with organic solvent or water. ACAT activity inhibitor. 4. The inhibitor of activity of ACAT according to claim 3, wherein the alcohol is selected from C ₁ to C ₄ alcohols and extracted. [4] The activity inhibitor of ACAT according to claim 3, wherein the organic solvent is selected from ethyl acetate, normal-hexane or chloroform and re-extracted. The preventive or therapeutic agent for cardiovascular disease caused by hypercholesterolemia containing the extract of claim 3 as an active ingredient.