KR100352269B1 - Lignan-type compound having antioxidant activity against a low density lipoprotein - Google Patents

Abstract

The present invention relates to a lignan compound having an antioxidant activity against a low density lipid protein, a method for preparing the same, and a pharmaceutical composition containing the same, specifically obtained by separating the compound from a lignan compound represented by the following formula (1) The present invention relates to a pharmaceutical composition for preventing and treating circulatory diseases including atherosclerosis associated with oxidation of low density lipid proteins and antioxidants for low density lipid proteins containing the compound as an active ingredient.

In the formula, R represents H, alkyl group or benzyl group.

Description

Lignan-type compound having antioxidant activity against a low density lipoprotein

The present invention relates to a lignan compound having an antioxidant activity against a low density lipid protein, a method for preparing the same, and a pharmaceutical composition containing the same, specifically obtained by separating the compound from a lignan compound represented by the following formula (1) The present invention relates to a pharmaceutical composition for preventing and treating circulatory diseases including atherosclerosis associated with oxidation of low density lipid proteins and antioxidants for low density lipid proteins containing the compound as an active ingredient.

Formula 1

In the formula, R represents H, alkyl group or benzyl group.

Atherosclerosis is an artery that thickens and hardens, loses its elasticity and weakens, and is one of the major diseases that occur with aging. Atherosclerosis is more likely to occur in the cerebral artery or coronary arteries. In the case of cerebral atherosclerosis, headache, dizziness, and mental disorders are indicated. It is known to become. In addition, this causes high blood pressure, heart disease, cerebral hemorrhage, and diseases caused by arteriosclerosis are emerging as the leading cause of death in modern society, especially among men in their 50s and 60s. Therefore, many studies have been conducted to elucidate the pathogenesis of atherosclerosis and to develop preventive and therapeutic agents.

There are many known causes of atherosclerosis, but among them, oxidized low density lipoprotein (Ox-LDL) produced by oxidizing low density lipoprotein (LDL) has been pointed out as the main cause of atherosclerosis. (Steinberg. D. et al ., N. Engl. J. Med. , 320 , 915-924 (1989)). Low-density lipid protein is one of the proteins that carries blood cholesterol. When it is oxidized, it does not function and is converted into small foam cells by macrophage in blood vessels, which infiltrate into vascular endothelial cells. Thicken smooth muscle. In addition, the oxidized lipid protein retains macrophages in the arterial wall, which leads to the formation of fatty glands on the vascular endothelium, thereby causing atherosclerosis.

On the other hand, three hundred seconds (Sarurus chinensis Baill) is a perennial herb of over three hundred years, and has been used in China since ancient times as an analgesic, diuretic and anti-inflammatory medicine. Since it was first discovered in Korea that three hundred seconds lived on Jeju Island about ten years ago, it has been used in the private sector for the treatment of cancer or chronic adult diseases such as circulatory disease, nervous system disease and diabetes.

Therefore, the inventors of the present invention, while searching for traditional Korean medicine and folk medicine to develop a new arteriosclerosis drug, found that the antioxidant activity of the low density lipid protein of 300 seconds is excellent and as an active ingredient, This H-lignan compound was isolated and found that the compound and its derivatives are very effective in inhibiting the oxidation of low density lipid proteins, thereby atherosclerosis associated with the oxidation of antioxidants and low density lipid proteins against low density lipid proteins. The present invention has been completed by elucidating the use of this compound as an agent for preventing or treating diseases of circulatory diseases.

It is an object of the present invention to provide a lignan compound having an antioxidant activity against a low density lipid protein, a preparation method thereof, and a pharmaceutical composition containing the same.

In order to achieve the above object, the present invention contains one or more of the lignan compound represented by the following formula (1) having an antioxidant activity against low density lipoprotein as an active ingredient, for the antioxidant for low density lipid protein It provides a pharmaceutical composition.

Formula 1

Wherein R represents H, an alkyl group or a benzyl group.

Preferably, in the present invention, in the low-density lipid protein containing one or more of the lignan-based compound having an antioxidant activity against the low-density lipid protein in which R is H, C ₁ -C ₆ alkyl group or benzyl group It provides a pharmaceutical composition for antioxidant.

More preferably, the lignan-based compound provides a pharmaceutical composition for antioxidant against low-density lipid protein, characterized in that R is H (machilin D) of the formula (2).

In another aspect, the present invention provides a pharmaceutical composition for antioxidant against low-density lipid protein, characterized in that the lignan compound is a virolin of the formula (3) wherein R is a methyl group.

In the lignan compound, M-linin (2), wherein R is H, may be obtained by separating from Saururus chinensis Baill.

Specifically, the present invention

1) extracting the 300 sec root powder with methanol (step 1);

2) extracting the extract of step 1 using water, n -hexane and chloroform (step 2);

3) separating the chloroform fraction of step 2 by silica gel column chromatography using a mixed solvent of n -hexane and ethyl acetate as a mobile phase (step 3);

4) separating the fraction of step 3 by reverse phase silica gel column chromatography using an aqueous methanol solution as the mobile phase (step 4);

5) separating and purifying the fraction of step 4 by silica gel column chromatography using a mixed solvent of chloroform and methanol as a mobile phase (step 5)

Provided is a method for preparing malin D.

In another aspect, the present invention provides a method for preparing a lignan compound of formula (1) characterized in that when R is an alkyl group in the formula (1), by changing the OH of the position 4 of the malinelin D to OR through an alkylation reaction.

The present invention also provides a pharmaceutical composition for preventing or treating circulatory diseases, including atherosclerosis associated with oxidation of low-density lipid protein, containing at least one of the compounds of Formula 1 as an active ingredient.

Hereinafter, the present invention will be described in detail.

In the present invention, machilyl D represented by the formula (2) is obtained by separating from 300 seconds by the following method.

The dried triticale root powder was extracted with methanol, concentrated, extracted with water and n -hexane to obtain a hexane layer, and the water layer was extracted with chloroform again to obtain a hexane layer, a water layer, and a chloroform layer. For each fraction, the antioxidant activity of the low density lipid protein was measured, and the chloroform layer showed the strongest antioxidant activity.

The chloroform layer is concentrated to give a dark brown oily substance, which is separated by silica gel column chromatography using a mixed solvent of n -hexane and ethyl acetate as a mobile phase. At this time, it is preferable to use a solvent having n -hexane: ethyl acetate = 90 to 10: 10 to 90 as the mobile phase.

This active fraction is separated again by reversed phase silica gel column chromatography using an aqueous methanol solution as the mobile phase. At this time, it is preferable to use 10-100% aqueous methanol solution as a mobile phase.

This active fraction is separated again by silica gel column chromatography using a mixed solvent of chloroform and methanol as a mobile phase. At this time, it is preferable to use a solvent having chloroform: methanol = 100 to 70: 0 to 30 as the mobile phase.

By the above method it is possible to obtain more than 12 mg of Machinin D per kg of dried 300 seconds sec.

In addition, when R is an alkyl group in the formula (1) it can be prepared by replacing the OH at position 4 of the malin D with an alkylation reaction to OR. At this time, the alkylation reagent may vary depending on the type of alkyl group to be prepared. Specifically, diazomethane (CH ₂ N ₂ ), potassium carbonate / ethyl iodide (K ₂ CO ₃ / Et-I), potassium carbonate / ethyl bromide (K ₂ CO ₃ / Et-Br), potassium carbonate / Preference is given to using propyl bromide (K ₂ CO ₃ / Pr-Br), potassium carbonate / benzyl bromide (K ₂ CO ₃ / Benzyl bromide). Moreover, it is preferable to use chloroform, diethyl ether, ethanol, acetone, dimethyl sulfoxide (DMSO) etc. as a reaction solvent.

On the other hand, in order to measure the antioxidant activity of the low density lipid protein of the compound of Formula 1, the low density lipid protein is extracted from the blood and Cu ²⁺ is added to induce oxidation. As a result, the machiline D and the viroline exhibited excellent antioxidant activity against low-density lipid proteins with IC ₅₀ of 2.8 μM and 4.5 μM, respectively. Antioxidant activity of malineline D and viroline has not been reported yet, and for the first time, the antioxidant activity of malineline D and viroline was revealed. Therefore, the compounds of Formula 1 according to the present invention can be used to prevent or treat circulatory diseases such as atherosclerosis, which are known to be caused by oxidation of low density lipid proteins.

The compounds according to the invention can also be used in the form of pharmaceutically acceptable salts, including all salts, hydrates and solvates prepared by conventional methods.

The compound according to the present invention can be administered orally or parenterally during clinical administration and can be used in the form of general pharmaceutical preparations.

The compound of the present invention may be administered in various oral and parenteral dosage forms during actual clinical administration, and when formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc., which are commonly used, may be used. It is prepared by. Solid preparations for oral administration include tablets, pills, powders, granules, capsules and the like, which solid preparations comprise at least one excipient such as starch, calcium carbonate, sucrose or lactose in at least one compound of formula (I). Mixed with gelatin. In addition to simple excipients, lubricants such as magnesium styrate talc are also used. Liquid preparations for oral administration include suspensions, solutions, 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. have. 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 a suppository base, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerol, gelatin, and the like may be used.

Dosage units may contain, for example, one, two, three or four times, or 1/2, 1/3 or 1/4 times the individual dosage. Individual dosages preferably contain an amount in which the active compound is administered at one time, which usually corresponds to a total, 1/2, 1/3 or 1/4 of the daily dose.

The effective dose of the compound of formula 1 is 0.1-100 mg / kg, preferably 0.5-10 mg / kg, and may be administered 1-3 times a day.

In addition, as a result of oral administration of the compound of the present invention to mice, 50% lethal dose (LD ₅₀ ) by oral toxicity test was found to be at least 1000 mg / kg or more.

Hereinafter, the present invention will be described in more detail with reference to Examples.

However, the following examples are illustrative of the present invention, and the content of the present invention is not limited by the examples.

Example 1 Isolation and Purification of Macillin D from 300 Seconds

2 kg of dried three hundred seconds (purchased in Geochang, Gyeongsangnam-do, Korea) were washed well, dried and ground using a grinder to form a powder. The white powder was added to 6 l of methanol and allowed to stand at room temperature for 24 hours. Then, the methanol solution was stirred and filtered through a filter paper to separate the liquid phase. The above methanol extraction process was repeated four times to collect only the liquid phase and concentrated under reduced pressure.

The methanol extract obtained above was suspended in 2 L of water, and 2 L of n -hexane was added thereto. The hexane layer was separated using a separatory funnel. The water layer was extracted again by adding chloroform, and the water layer and the chloroform layer were separated using a separatory funnel.

The hexane layer (25 g), the chloroform layer (20 g) and the water layer (150 g) obtained in the above process were measured for antioxidant activity against low density lipid protein, respectively. Antioxidant activity of each fraction was measured by the method of the following experimental example.

Experimental results showed the strongest antioxidant activity in the chloroform layer, the chloroform layer was concentrated and dried under reduced pressure to give a dark brown oily (oily) material. Chloroform extract was separated and purified by the following method again.

The chloroform extract obtained above was separated by silica gel column chromatography (silica gel: Merck, Art 9385, column size: Ø7.5 x 45 cm) with varying ratios of n -hexane and ethyl acetate. As a result, the active material was most effectively separated using a solvent with n -hexane: ethyl acetate = 60:40, whereby 2 g of the active material were obtained.

The active fraction was separated by C-18 (Merck, Lichroprep RP-18, Art 13900) column chromatography using a 70% aqueous methanol solution as a mobile phase (column size: Ø4.2 x 29 cm) to obtain 1 g of the active fraction. Got it.

The active fractions were subjected to silica gel column chromatography (column size: Ø2.6 x 25 cm) with varying ratios of chloroform and methanol. In the case of using a solvent of chloroform: methanol = 98: 2, the active substance was most effectively separated. At this time, 24 mg of malineline D, a pure active substance, was obtained.

Example 2 Preparation of Virollin

5 mg of Methylin D was added to a small glass bottle, and 0.3 mg of chloroform was added thereto to dissolve. 1 ml of etheric diazomethane was added thereto, sealed, and left to stand at room temperature for 12 hours. After 12 hours, the solvent was removed under reduced pressure and purified by silica gel column chromatography (solvent: chloroform: methanol = 98: 2) to obtain 4.5 mg of viroline (yield: 90%).

Example 3 Structural Analysis of Macillin D and Virollin

The materials obtained through Examples 1 and 2 were determined using a VG high resolution GC / MS spectrometer, Election Ionization MS, Autospec-Ultima, and the molecular weight and molecular formula were determined. Measurement was made using a polarizer (Jasco DIP-181 digital polarimeter). In addition, ¹ H NMR, ¹³ C NMR, Homo-Cozy, HMQC ( ¹ H -Detected heteronuclear M ultiple- Q uantum C oherence through nuclear magnetic resonance (NMR) analysis (Bruker DMX 600, Bruker AMX 500) ), HMBC (Heteronuclear Multiple-Bond Coherence), DEPT (Distortionless Enhancement by Polarization) spectra were obtained, and molecular structure was determined. The measurement results are as follows. As a result of comparative analysis with the published literature, the compound of formula (2) was used as Methylin D (Shimomura, H., Sashida, Y. and Oohara, M. Phytochemistry 26 : 1513-1515, 1987). As a result, the compound of formula 3 was identified as virolin (Zacchino, SA J. Nat. Prod. 57 : 446-451, 1994).

Machilin D

1) Physical property: Light brown amorphous powder

2) Radiance: [α] _D ²⁵ -88 (c = 4, CHCl ₃ )

3) Molecular Weight: 344

4) Molecular Formula: C ₂₀ H ₂₄ O ₅

5) ¹ H-NMR (CDCl ₃ , 500 MHz): see Table 1

6) ¹³ C-NMR (CDCl ₃ , 125 MHz): see Table 1

NMR Results of Macillin D location ¹ H (integral value) multiplicity ( J ) ¹³ C One 132.6 One' 134.1 2 6.84 (1 H) d (1.8) 110.2 2' 6.85 (1 H) d (2.0) 109.9 3-OCH ₃ 3.78 (3 H) s 56.6 3 147.4 3 ' 151.4 4-OCH ₃ 3.82 (3 H) s 56.8 4 146.2 4-OH 5.66 (1 H) s 4' 147.3 5 6.86 (1 H) d (8.2) 114.8 5 ' 6.80 (1 H) d (8.2) 119.6 6 6.78 (1 H) dd (1.8, 8.2) 121.4 6 ' 6.79 (1 H) dd (2.0, 8.2) 119.4 7 4.53 (1 H) d (8.3) 79.1 7-OH 4.12 (1 H) bs 7 ' 6.27 (1 H) d (1.5, 15.7) 131.3 8 4.02 (1 H) dq (6.3, 8.3) 84.8 8' 6.06 (1 H) dq (6.6, 15.7) 125.5 9 1.07 (3 H) d (6.3) 17.7 9 ' 1.79 (3 H) d (1.5, 6.6) 19.0

Viroline

1) Physical property: Light brown amorphous powder

2) Luminous intensity: [α] _D ²⁵ -32 (c = 1, CHCl ₃ )

3) Molecular Weight: 356

4) Molecular Formula: C ₂₁ H ₂₆ O ₅

5) ¹ H-NMR (CDCl ₃ , 500 MHz): see Table 2

6) ¹³ C-NMR (CDCl ₃ , 125 MHz): see Table 2

NMR Results of Virollin Atom ¹ H (integral value) multiplicity ( J ) ¹³ C One 132.5 One' 146.7 2 6.86 (1 H) 110.0 2' 150.8 2'-OCH ₃ 3.85 (3 H) s 55.7 3 149.0 3 ' 6.85 (1 H) 109.2 3-OCH ₃ 3.82 (3 H) s 56.9 4 148.8 4' 133.5 4-OCH ₃ 3.81 (3H) s 56.9 5 6.76 (1 H) d (8.2) 110.8 5 ' 6.79 (1 H) dd (1.8, 8.2) 118.8 6 6.84 (1 H) dd (1.9, 8.2) 120.0 6 ' 6.87 (1 H) d (8.2) 119.0 7 4.56 (1 H) d (8.4) 78.4 7 ' 6.28 (1 H) d (1.4, 15.8) 130.4 7-OH bs 8 4.02 (1 H) dq (6.3, 8.3) 84.2 8' 6.08 (1 H) dq (6.6, 15.8) 124.9 9 1.09 (3H) d (6.2) 17.0 9 ' 1.80 (3 H) d (1.4, 6.6) 18.3

Experimental Example Antioxidant Activity against Low Density Lipid Protein

Through the following experiments, the antioxidant activity of the low density lipid protein of the compound of Formula 1 isolated in three hundred seconds was investigated.

Cu ²⁺ is known to induce oxidation of low density lipid proteins (Cu ²⁺ mediated LDL-oxidation). Therefore, in the present invention, dialdehyde (dialdehyde), which is an oxidation product of the unsaturated fatty acid produced at this time, was measured by TBA (thiobarbituric acid) method, and the antioxidant activity of the compound of Formula 1 was investigated (Packer, L. Ed. (1994) Methods in Enzymology Vol. 234, Oxygen radicals in biological systems Part D. Academic press, San Diego).

300 ml of human plasma was centrifuged at 100,000 × g for 24 hours using an ultracentrifuge to remove the high-density lipid protein (VLDL) / kilomicron layer suspended in the upper layer and the specific gravity of the remaining solution to 1.063 g / ml. After aligning, 35 ml (1.5-2.5 mg protein / ml) of low-density lipid protein suspended in the upper layer was again separated by centrifugation at 100,000 × g for 24 hours. 50 μl of the low-density lipid protein thus obtained (50-100 μg of protein) was mixed with 180 μl of 10 mM phosphate-buffered saline (PBS), and 10 μl of lignan compound solution prepared at various concentrations was added. . Machilin D and viroline were dissolved in DMSO (dimethylsulfoxide), respectively, and diluted to various concentrations before use in experiments. As the control, only the solvent was added.

10 μl of 0.25 mM CuSO ₄ was added to the solution and reacted at 37 ° C. for 2 hours. The reaction was stopped by adding 1 ml of 20% trichloroacetic acid (TCA) solution. 1 ml of a 0.67% TBA solution dissolved in 0.05 N NaOH solution was added thereto, stirred for 10 seconds, and heated at 95 ° C. for 5 minutes to cause a color reaction. The solution was cooled with ice water. The solution was centrifuged at 3000 rpm for 5 minutes to separate the supernatant, and the absorbance at 540 nm was measured by UV-vis spectroscopy to determine the amount of malondialdehyde (MDA) produced by the color reaction. .

Meanwhile, using a stock solution of tetramethoxypropane (malonaldehyde bis (dimethylacetal)), 250 µl of PBS standard solution containing 0-5 nmol malondialdehyde was prepared. The standard solution was developed in the same manner as described above, and the absorbance at 540 nm was measured, and the standard curve of malondialdehyde was determined, and the amount of malonaldehyde was quantified using the standard curve in the above experiment using the compound of Formula 1. .

Antioxidant activity of the low density lipid protein of the compound of Formula 1 is shown in Table 3 below.

Antioxidant Activity of Low Density Lipid Proteins of Compounds of Formula 1 compound IC ₅₀ (μM) Machilin D 2.8 Viroline 4.5

As can be seen in Table 3 , the lignan-based compounds of the present invention, mashin D and virollin IC ₅₀ was very low as 2.8 μM, 4.5 μM, respectively, it was found that the antioxidant activity against low-density lipid protein is excellent. Therefore, the compound of Formula 1 according to the present invention can be usefully used as a prophylactic or therapeutic agent for circulatory diseases such as arteriosclerosis, which is known to be caused by oxidation of low density lipid protein.

As described above, since the lignan compound of the present invention, the compound of formula 1, in particular, the mashin D and viroline is excellent in preventing the oxidation of low-density lipid protein, low-density lipid protein, including antioxidants for low-density lipid protein It may be useful for the purpose of preventing or treating circulatory diseases such as atherosclerosis known to be caused by oxidation.

Claims (5)

Pharmaceuticals useful for the prevention and treatment of atherosclerosis associated with oxidation of low density lipid proteins, containing as an active ingredient one or more of the lignan compounds represented by the following formula 1 having antioxidant activity against low density lipoproteins: Composition. Formula 1 In the formula, R represents hydrogen or methyl group. The pharmaceutical composition according to claim 1, wherein the lignan compound is machilin D of Formula 2 wherein R is H. Formula 2 According to claim 1, wherein the lignan compound is a pharmaceutical composition, characterized in that R is a methyl group (virolin) of the formula (3). Formula 3 3. The pharmaceutical composition according to claim 2, wherein the mashin D of Formula 2 wherein R is H is obtained separately from Saururus chinensis Baill. delete