KR20120082047A - A composition comprising obacunone for treating or preventing vascular disease - Google Patents

Abstract

PURPOSE: A composition containing obacunone is provided to suppress activation of arginase, enhance NO generation, and to treat hypertension, arteriosclerosis, cerebrovascular diseases, peripheral vascular diseases, or aortic disease. CONSTITUTION: A pharmaceutical composition for preventing or treating vascular diseases contains obacunone of chemical formula 1 as an active ingredient. The vascular diseases include arteriosclerosis, hypertension, coronary artery disease, cerebrovascular disease, peripheral vascular disease or artery disease. A health functional food for preventing or treating vascular diseases contains obacunone. The health functional food includes drink, alcoholic drink, or vitamin complex.

Description

A composition comprising obacunone for treating or preventing vascular disease

The present invention relates to a composition for the treatment or prevention of vascular diseases containing obecunone (obacunone).

More specifically, the present invention inhibits the activity of arginase and promotes the production of nitric oxide (NO) to relax vascular endothelial cells, thereby atherosclerosis, hypertension, coronary artery disease, cerebrovascular disease, The present invention relates to a composition for the prevention or treatment of vascular diseases containing obubenon that can treat peripheral vascular disease or aortic disease.

Endothelial vessels play an important role in maintaining blood vessel homeostasis for vasoreactivity, platelet activity, leukocyte adhesion, smooth muscle cell proliferation and migration.

NO (nitric oxide), which is produced in the vascular endothelium, is the most potent substance that protects blood vessels, and relaxes vascular smooth muscle cells to expand blood vessels. Damaged NO signaling system leads to atherosclerosis and hypertension. Resulting in vascular diseases such as. NOS (nitric oxide synthase) is, O _2, and co-factor nicotinamide adenine dinucleotide phosphate (nicotinamide adenine dinucleotide phospate, NADPH) , flavin adenine dinucleotide (flavin adenine dinucleotide, FAD), flavin mono nucleotide (flavin mononucleotide, FMN ), A group of enzymes that synthesize NO from the terminal nitrogen atom of L-arginine in the presence of heme, tetrahydrobiopterin (BH4), and among the known NOSs, n-NOS (neuronal nitric) Oxide synthase), e-NOS (endothelial NOS), and i-NOS (inducible NOS) are three types, among which e-NOS produces NO and is involved in vascular function regulation.

Arginase, on the other hand, is an enzyme that degrades L-arginine, a substrate, and has a competitive relationship with e-NOS, which is known as an enzyme capable of regulating NO production (Morris et. al ., 1998; Berkowitz et al ., 2003; Simon et al ., 2003; Holowatz et al ., 2006; Steppan et al ., 2006; Peyton et al ., 2009). Arginase also modulates NOS activity by limiting the bioavailability of L-arginine (Ryoo et al. al ., 2006), the inhibition of arginase actively increases NO production and is effective for normal heart function, as well as atherosclerosis, aging, erectile dysfunction, sickle cell anemia, which are abnormal blood vessels (sickle cell disease) has been reported to be effective (Bivalacqua et. al ., 2001; Berkowitz et al ., 2003; Morris et al ., 2004; Steppan et al ., 2006; White et al ., 2006; Bivalacqua et al ., 2007; Hsu et al ., 2007; Xu et al ., 2007).

Inhibition of arginase is associated with the aortic endothelium of the redd (Berkowitz et al. al ., 2003), Bovine Lung Endothelial Cells (Chicoine et. al ., 2004), coronary arteries of pigs (Zhang et al ., 2001), etc., showed that NO production was prominent and also inhibited the proliferation of human umbilical vein endothelial cells (HUVECs) and was also associated with the inhibition of angiogenesis (Faffe et al. al ., 2005).

Arginase currently has two isomers. The expression and function of arginase I in macrophages, hepatocytes, and vascular smooth muscle cells can be expressed by LPS, interleukin-13, altered oxygen tension, and coronary artery swelling. It is known to be stimulated by balloon dilatation of coronary arteries (Modolell et. al ., 1995; Louis et al ., 1998; Que et al 1998; Klasen et al ., 2001; Chicoine et al ., 2004; Morris et al ., 2004; Ryoo et al ., 2006; Nelin et al ., 2007). The activity and expression of arginase II in the vascular endothelium is characterized by oxidized low-density lipoprotein (OXLDL), lipopolysaccharide (LPS), tumor necrosis factor-α (TNF-α), interferon-β (IFN-β), and 8-Bromo. -cGMP (8-Bromoguanosine 3 ', 5'-cyclic monophosphate) and hypoxia are known to be induced by various blood vessel injuries (Morris et. al ., 1998; Que et al ., 1998; Chicoine et al ., 2004; Ryoo et al ., 2006; Nelin et al ., 2007).

In addition, high arginase II activity in atherosclerosis-prone mice also impairs vascular endothelial NO production, endothelial dysfunction. It has been shown to be associated with vascular stiffness and, in particular, aortic plaque development in the aorta. In contrast, inhibition of endothelial arginase or deletion of arginase II gene increased NO production, vascular endothelial and aortic function was restored, and plaque production was reduced. Therefore, arginase II is a major target for the prevention or treatment of vascular diseases including atherosclerosis (Ryoo et al. al . , 2008), increased arginase activity has been shown to affect endothelial dysfunction, systemic and pulmonary hypertension.

Ovequinone, a compound of the present invention, is known to have an active effect on the central nervous system and appetite (European Patent No. 498890), and a limonoid compound containing obequinone is used to treat neurodegenerative disease. It can also be known (J Mol Neurosci. 2010, 42 [1], 9-16). In addition, limonoid compounds containing obubenon have anti-proliferative effects on neuronal and colon cancer cells (Nutr Cancer. 2006, 56 [1], 103-12), and drug-sensitive cells. In addition, it is known to have a cytotoxic effect similar to vincristine, vinblastine and taxol on multidrug-resistant cells, and is also expected to be used as an anticancer agent (J Agric Food Chem). 2004, 52 [15], 4908-12). In addition, extracts of red mexican grapefruit seeds containing obekunon are known to have antioxidant effects. Obekunon has various physiological activities (Z Naturforsch C. 2007, 62 [3-4]). ], 179-188).

However, it has not yet been found that obekunon has a therapeutic effect on vascular diseases such as atherosclerosis and hypertension. On the other hand, the present inventors confirmed that the obekunon inhibits arginase activity and increases NO production, and revealed that the compound of the present invention can be used as a therapeutic agent for vascular diseases such as atherosclerosis and hypertension. The invention has been completed.

The present invention is to provide a composition for the treatment or prevention of vascular diseases containing obekunone.

In the present invention, it is confirmed that the obekunone represented by the following Chemical Formula 1 inhibits the activity of arginase and activates the production of nitric oxide (NO) in the vascular endothelium, and the obekunone is used as a therapeutic agent for vascular diseases. The present invention has been completed by revealing that it can be used.

[Formula 1]

Obekunon, a compound of the present invention, can be extracted from sulfur white (Huang, dried bark of the rhubarb belonging to the rutaceae), water, ethanol, methanol, butanol, propanol, acetone, ethyl acetate, methylene chloride , Extraction with hexane or a mixed solvent thereof, distribution of an organic solvent and water, a method by column chromatography, a known method used for separate extraction of plant components, and the like can be easily obtained by combining them alone or suitably. The crude extract can be further purified according to a commercial method as needed.

Compound of the present invention represented by Formula 1 Obecuun can be prepared as a pharmaceutically acceptable salt according to a conventional method in the art.

In another aspect, the present invention is a pharmaceutical composition for the prevention and treatment of vascular diseases, such as atherosclerosis, hypertension, coronary artery disease, cerebrovascular disease, peripheral vascular disease and aortic disease, containing obekunon or a pharmaceutically acceptable salt thereof as an active ingredient. To provide.

Pharmaceutical compositions for the prevention and treatment of vascular diseases comprising obekunone or a pharmaceutically acceptable salt thereof according to the present invention, powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, respectively, according to conventional methods Oral dosage forms, external preparations, suppositories, and sterile injectable solutions. Carriers, excipients, and diluents that may be included in the composition comprising the obbecuone or a pharmaceutically acceptable salt thereof include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber , Alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil have. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations may contain at least one excipient such as starch, calcium carbonate, or the like in the obecuone or a pharmaceutically acceptable salt thereof. , Sucrose or lactose, gelatin and the like are mixed. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The dosage of the obubecunon or a pharmaceutically acceptable salt thereof will vary depending on the age, sex and weight of the subject to be treated, the specific disease or pathology to be treated, the severity of the disease or pathology, the route of administration and the judgment of the prescriber. Dosage determination based on these factors is within the level of skill in the art and generally dosages range from 0.01 mg / kg / day to approximately 2000 mg / kg / day. A more preferable dosage is 0.1 mg / kg / day to 500 mg / kg / day. Administration may be once a day or may be divided several times. The dose is not intended to limit the scope of the invention in any way. The obubequinone of the present invention or a pharmaceutically acceptable salt thereof can be administered to mammals such as rats, livestock, humans, etc. by various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine dural or intracerebral injection. In addition, the compound of the present invention obekunon is a compound derived from natural products extracted from sulfur white, so there is almost no toxicity and side effects, it is a drug that can be used safely even for long-term administration for the purpose of prevention.

The present invention also provides a dietary supplement for the prevention and improvement of heart disease, including the compound Obecunon and food supplements acceptable food supplement. The health functional food of the present invention includes tablets, capsules, pills or liquids, and the like, and the health functional foods include dairy products including soups, meat, sausages, breads, candy, snacks, noodles, ice cream, soups, Beverages containing ionic beverages, nutritional products including alcoholic beverages and vitamin complexes may be included.

The present invention relates to a composition for the prevention and treatment of vascular diseases containing obekunon, the treatment of the obekunon, it was confirmed that the activity of arginase is suppressed, the production of NO in the aorta increases. . Inhibition of arginase activity and increased production of NO in vascular cells are known to have a therapeutic effect on hypertension or atherosclerosis. Thus, the obekunone compound of the present invention is hypertension, arteriosclerosis, coronary artery disease, cerebrovascular disease. It has been confirmed that vascular diseases such as peripheral vascular disease or aortic disease can be treated.

1 is a graph showing concentration-dependent inhibition of the activity of arginase II in the kidneys of mice due to the treatment of obbecuone.
FIG. 2 is a graph showing concentration-dependent inhibition of the activity of arginase I in the liver of mice due to the treatment of obbecuone.
Figure 3 is a graph showing that the production of NO in the mouse aorta due to the treatment of obekunon.

Hereinafter, a preferred embodiment of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein but may be embodied in other forms. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the concept of the invention to those skilled in the art.

< Example  One. Obekunon  Separation / purification / and analysis of physical and chemical properties and chemical structure>

6 kg of sulfur white was added to 10 l of a mixture of methanol and methylene chloride in a ratio of 1: 1, and extracted three times at room temperature for 5 days. The extracts were collected and concentrated under reduced pressure to obtain an extract (898 g). The extract was suspended in 5 L of distilled water and the same amount of methylene chloride was added. The methylene chloride fraction and water fraction were fractionated several times using a separatory funnel, and only the methylene chloride fraction was collected and concentrated under reduced pressure to obtain 519 g of methylene chloride fraction. N -butanol was added in the same manner to obtain 169 g of n -butanol fraction.

136 g of methylene chloride fractions were fractionated by column chromatography as follows. Fill the column with a diameter of 9cm with silica gel 80cm, n -hexane flow out to make the fixed phase homogeneous, and after filling the sample, the ratio of ethyl acetate to n -hexane starts from 0% to 2% unit. Fraction 1 to Fraction 16 was obtained by distilling the mobile phase with a gradient elution increasing to 5% starting at 20% and 10% starting at 50%.

The fraction 13 (11.9g) was filled with 50cm silica gel in a column having a diameter of 5cm and methylene chloride was flowed out to make the stationary phase uniform and filled with a sample. After saturating the column with methylene chloride, the mobile phase was distilled off by gradient elution increasing the ratio of acetone to methylene chloride by 3% to obtain a small fraction 13.6 in small fraction 13.1. Among them, a small fraction 13.1 (4.1 g) was recrystallized from methanol to obtain obecunone (2,100 mg). The chemical structure of the compound was analyzed based on the results of ¹ H and ¹³ C-NMR analysis of nuclear magnetic resonance analysis, and the structure was determined by comparison with the literature (Chem. Pharm. Bull, 36 [11], 4453). -4461; Arch Pharm Res, 30 [1], 58-63; Arch Pharm Res, 30 [1], 58-63; Bio Factors, 13, 213-218)

Gone into kyunon (Obacunone): colorless crystal; mp 228-235 ℃. -46.4 ° (c = 0.24, CHCl ₃ ); IR (neat) V _max : 2988, 1745, 1703, 1630, 1572, 1503, 1451, 1393, 1282 cm ^-1 . EI-MS m / z (rel. Int.): 454 [M] ⁺ (0.4), 439 (4), 397 (3), 363 (15), 347 (2), 331 (100), 313 (7 ). ¹ H-NMR (250 MHz, CDCl ₃ ) δ 7.43 (1H, s, H-21), 7.40 (1H, s, H-23), 6.53 (1H, d, J = 11.7 Hz, H-1), 6.37 (1H, s, H-22), 5.97 (1H, d, J = 11.0 Hz, H-2), 5.46 (1H, s, H-17), 3.66 (1H, s, H-15), 2.99 (1H, t, J = 11.0 Hz, H-6b), 2.61 (1H, dd, J = 11.0 Hz, J = 4.0 Hz, H-5), 2.30 (1H, dd, J = 11.0 Hz, J = 4.0 Hz, H-6a), 2.15 (1H, dd, J = 8.5 Hz, J = 3.5 Hz, H-9), 1.50 (6H, s, H-29, H-30), 1.46 (3H, s, H -28), 1.24 (3H, s, H-19), 1.12 (3H, s, H-18); ¹³ C-NMR (63 MHz, CDCl ₃ ): δ 207.4 (C-7), 166.9 (C-3), 166.7 (C-16), 156.8 (C-1), 143.3 (C-23), 140.9 ( C-21), 122.8 (C-2), 120.0 (C-20), 109.7 (C-22), 84.0 (C-4), 78.0 (C-17), 65.0 (C-14), 57.2 (C -5), 53.4 (C-15), 53.1 (C-8), 49.1 (C-9), 43.0 (C-10), 39.8 (C-6), 37.3 (C-13), 32.6 (C- 12), 31.9 (C-30), 26.7 (C-19), 21.0 (C-29), 19.4 (C-28), 16.9 (C-11), 16.4 (C-18).

< Example  2. Obekunon  Confirmation of Arginase Inhibitory Effect>

Arginase activity assays are described in Ryoo et. al . (CircRes 2006, 99, 951-960).

To this end, cell lysis buffers (50 mM Tris-HCl, pH 7.5, 0.1 mM EDTA and protease inhibitors) were treated and homogenized at 4 ° C in the liver and kidney of C57BL / 6 mice, centrifuged at 14,000 x g, and only the supernatant was taken. Arginase lysate was obtained. The arginase lysate was diluted 10-fold with 50 mM tris-HCl, 33.5 µl of 50 mM Tris-HC, 1.5 µl of obecuone (1 mg / µl), 25 µl of 10-fold diluted arginase lysate, L-arginine 25 μl (L-arginine, 0.1M) was added. Thereafter, the reaction was carried out at 37 ° C. for 1 hour, and then the reaction was stopped with an acid solution (H ₂ SO ₄ : H ₃ PO ₄ : DW = 1: 3: 7). Next, 25 μl of INSP (9% α-isonitrosopropiophenone in absolute ethanol) was added and color developed at 97 ° C. for 45 minutes (color reaction with urea). After 10 minutes of dark reaction, absorbance was measured at 550 nm wavelength. In addition, the absorbance values were compared with the control without obekunon treatment. The enzyme activity (pmol Urea / min / mg protein) was calculated from the urea standard curve, and the inhibition of enzyme activity was determined by measuring arginase activity in the presence of the obbecuone compound and thus the percentage of enzyme activity in the absence of the obbecuone compound. Marked as.

Arginase is an isoform of arginase I and II, mainly expressing arginase I in the liver and arginase II in the kidney, and has already been confirmed by Western blot analysis (Experimental and molecular medicine, 42 [7], 524-532, 2010). In the present invention, the activity of arginase I in the liver and the activity of arginase II in the kidney were confirmed. As a control of this experiment, a liver and kidney lysate obtained by treating DMSO (dimethylsulfoxide), which is a solvent of the obekunone compound, in the same volume as the amount of the obekundone was used.

Referring to Figures 1 and 2, it was confirmed that obekunon inhibited the activity of arginase II in the kidney by concentration, respectively, and inhibited the activity of arginase I in the liver. As a result, the present invention It was able to prove that the obubequinone compound had an effect of inhibiting both arginase I and II.

< Example  3. Obekunon NO  Check the creation effect>

Measurement of NO was confirmed by nitrogen monoxide production test using DAF-AM (4-Amino-5-methylamino-2 ', 7'-difluorofluorescein diacetate), a NO-sensitive fluorescent dye in aorta isolated from mice. .

After aortic rings were isolated from mice, the culture medium (Dulbecco's modified Eagle's medium with 2% FBS and antibiotics [1 ×]) was incubated for at least 18 hours with 30 μg / ml of Obecuone. It was.

To confirm NO production, the aorta of the mouse was cut vertically and subjected to HEFES buffer (NaCl 120 mM, KH ₂ PO ₄ 2.6 mM, KCl 4 mM, CaCl _{2 2} mM, MgCl ₂ 0.6 mM, HEPES 25 mM, glucose 14 mM, pH7.4). It was plugged into the bottom of a silgard-coated chamber. The chamber was then kept at 37 ° C. and the medium condition was kept constant (static bath conditions).

The degree of NO production in aortic tissues was measured by fluorescence using DAF-FM (measurement conditions → Olympus 10 × objective with optimized excitation, emission wavelength: [DAF-FM, 470/525 nm; or DHE, 470/580). ], intensified camera: [Luca 658M-TL], custom image acquisition program: [Cell software, Olympus]). First, the background fluorescence was measured and recorded at the first equilibrium. After leaving the aorta at room temperature for 15 minutes, 5 μM DAF-FM was treated for 10 minutes with HEPES in the aorta. After that, DAF-FM was removed, the fluorescence was measured and recorded, and the aorta was placed at 37 ° C. for 20 minutes. After removal of DAF-FM, L-NAME (N- ^g- nitro-L-arginine methyl ester, NOS inhibitors, 10 ^-5 μM) was treated to determine the difference in fluorescence level after the Origin (version 7.5, OrignLab Corp, Northampton) , MA). As a control of this experiment, a mouse aorta in which DMSO (dimethylsulfoxide), a solvent of the obubequinone compound, was treated in the same volume as the amount of the obevequinone was used.

The results of the analysis are shown in FIG. 3, and the results of FIG. 3 confirm that the obekunon significantly increases the production of NO in the mouse aorta.

< Example  4. Toxicity Test>

4-1. Acute Toxicity

This experiment was conducted to investigate the toxicity of animal animals acutely (within 24 hours) when overdose of obecunone was taken in a short period of time and to determine the mortality rate. Twenty ICR mouse strains, which are common mice, were assigned to 10 control groups and 10 experimental groups. No control group was administered, and the experimental group orally administered obekunon at a concentration of 2.0 g / kg (about 50 times the amount used in general animal experiments). The mortality was examined 24 hours after administration, and both the control group and the experimental group to which 2.0 g / kg of Obecuone was administered survived.

4-2. Long-term and tissue toxicity experiments in experimental and control groups

In order to investigate the effects on the organs (tissues) of C57BL / 6J mice, blood was collected after 8 weeks from the animals of the experimental group administered with Ovecuone and the control group administered with solvent only. GPT (glutamate-pyruvate transferase) ) And blood concentrations of Blood Urea Nitrogen (BUN) were measured using a Select E (Vital Scientific NV, Netherland) instrument. As a result, in the case of GPT, which is known to be related to hepatotoxicity, and BUN, which is known to be related to nephrotoxicity, there was no difference in the experimental group compared to the control group. In addition, liver and kidneys were excised from each animal, and histological observations were performed by optical microscopy through a conventional tissue fabrication process. No abnormalities were observed.

< Examples  1. Pharmaceutical Formulation example >

1-1. Manufacture of tablets

20 g of the compound obekunon of the present invention was mixed with 175.9 g of lactose, 180 g of potato starch, and 32 g of colloidal silicic acid. 10% gelatin solution was added to the mixture, which was then ground and passed through a 14 mesh sieve. It was dried and the mixture obtained by adding 160 g of potato starch, 50 g of talc and 5 g of magnesium stearate was made into a tablet.

1-2. Preparation of Injection

2 g of the compound Obecunon of the present invention was dissolved in distilled water to make 100 ml. The solution was bottled and sterilized by heating at 20 ° C. for 30 minutes.

< Examples  2. Food Manufacturing example >

2-1. Preparation of Cooking Seasonings

0.2 to 10% by weight of the compound obekunon of the present invention was prepared for cooking spices for health promotion.

2-2. Manufacture of flour food products

The compound of the present invention is added to flour with 0.1 to 5.0% by weight of the obekunon, and using the mixture to prepare bread, cakes, cookies, crackers and noodles to prepare a health-promoting food.

2-3. soup  And juicy ( gravies Manufacturing

Compound Obecunon of the present invention was added to the soup and broth with 0.1 to 1.0% by weight to prepare meat products for health promotion, soup and noodles of noodles.

2-4. dairy product( dairy products Manufacturing

Compound Obecunon of the present invention was added to milk at 0.1 to 1.0% by weight, and various dairy products such as butter and ice cream were prepared using the milk.

< Examples  3. Drink Manufacturing example >

3-1. Vegetable juice  Produce

0.5 g of the compound Obecunon of the present invention was added to 1,000 ml of tomato or carrot juice to prepare vegetable juice for health promotion.

3-2. Fruit juice  Produce

0.1 g of the compound Obecunon of the present invention was added to 1,000 ml of apple or grape juice to prepare a fruit juice for health promotion.

Claims (4)

A pharmaceutical composition for the prevention or treatment of vascular diseases containing obekunone of Formula 1 as an active ingredient.
[Formula 1]

The method of claim 1,
The vascular disease is a pharmaceutical composition for preventing or treating vascular diseases, characterized in that selected from the group consisting of arteriosclerosis, hypertension, coronary artery disease, cerebrovascular disease, peripheral vascular disease and aortic disease. Health functional food for the prevention or improvement of vascular diseases, characterized in that it contains obekunone (obacunone). The method of claim 3, wherein
The health functional food is selected from the group consisting of nutritional products including drinks, meat, sausages, bread, candy, snacks, noodles, dairy products including ice cream, soups, beverages including ionic drinks, alcoholic beverages and vitamin complexes Functional food for the prevention or improvement of vascular diseases, characterized in that.

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