KR100704003B1 - A compound comprising 2?-hydroxy-oleanolic acid for prevention and control of thrombosis - Google Patents

Abstract

The present invention relates to a composition for preventing and treating thrombin-inhibited thrombosis containing 2-alpha-hydroxy-oleanolic acid, cloves, jujube, bokbunja, olive tree, Chinese quince, loquat tree, loquat leaf 2-alpha-hydroxy-oleanolic acid (2α-hydroxy-oleanoic acid), which is separated and purified through various solvents such as water, alcohol, methanol, ethanol, butanol, sequential organic solvent fractions and silica gel chromatography It has an excellent effect on providing oleanolic acid. In addition, the present invention 2-alpha-hydroxy-oleanolic acid (2α-hydroxy-oleanolic acid) exhibits excellent thrombin inhibitory activity, can effectively suppress the formation of thrombus, and through thrombosis, thrombosis such as ischemic stroke and hemorrhagic stroke It can be used for the prevention and treatment of In addition, it is isolated from the cloves and loquat leaves that have been used for food, and has excellent safety. I can prepare it.

2-alpha-hydroxy-olenolic acid, thrombin inhibitors, hematogenous improvement, antithrombotic activity

Description

A compound comprising 2α-hydroxy-oleanolic acid for prevention and control of thrombosis}

1 shows the structure of a thrombin inhibitory activator isolated from cloves.

The present invention relates to a composition for preventing and treating thrombin-inhibited thrombosis containing 2-alpha-hydroxy-oleanolic acid. More specifically, the triterpenoid compound through water or organic solvent extraction, sequential organic solvent fraction and silica gel chromatography step of any of clove, jujube, bokbunja, olive, Chinese quince, loquat, loquat or Chinese hydrangea. Phosphoryl 2-alpha-hydroxy-oleanolic acid was isolated and its thrombin inhibitory activity, antithrombotic activity, thermal stability, acid stability and plasma stability were investigated to prevent oral thrombin inhibited thrombosis and It relates to providing a use as a therapeutic composition.

As a biological component, blood has a variety of important functions such as transporting and buffering oxygen, nutrients, and waste products, maintaining body temperature, controlling osmotic pressure and ion balance, maintaining moisture, controlling fluid, maintaining and controlling blood pressure, and protecting the body. have. Therefore, the functional abnormality of the blood causes a variety of diseases, in particular, cerebrovascular disease or heart disease resulting from abnormal hemostatic function to minimize blood loss in the damaged blood vessels are fatal to life and serious sequelae. At present, cardiovascular disease is rapidly increasing with the development of society and the aging of the population, and the sum of the mortality rates is higher than that of malignant tumors, and the development of effective prevention and treatment is urgently needed.

Normal blood circulation facilitates blood circulation as the blood coagulation reaction system and the thrombolytic reaction system in the body complement each other, and among them, the mechanism of the blood coagulation reaction system forms platelet thrombus by adhering and agglomerating platelets to blood vessel walls. In addition, it has been reported that the blood coagulation system is activated to form fibrin clots around platelet aggregates. The production of fibrin thrombi is a multistep reaction of numerous factors that activates thrombin, which is directly involved in fibrin coagulation, and finally produces fibrin monomers from fibrinogen, which are polymerized by calcium, binding to platelets and endothelial cells. Persistent thrombus formation is achieved by forming fibrin polymers cross-linked by Factor XIII. In addition, thrombin plays a pivotal role in thrombus formation by activating platelets, factor V and factor VII to promote blood coagulation. Therefore, thrombin activity inhibitors can be used as a very useful prophylactic and therapeutic agent for various thrombotic diseases caused by excessive blood clotting disorders, and aims to develop thrombin direct inhibitors worldwide. To date, heparin, coumarin, aspirin, urokinase and other anticoagulants, antiplatelets, and thrombolytics have been used for the prevention and treatment of thrombotic diseases. Its use is limited to such circumstances.

On the other hand, Clove ( Syzygium aromaticum ) is an evergreen sub-tree belonging to the plated cactus, which is cultivated to obtain spices in the Molucca Islands and the tropics, and dried clove is an important spice in the world. Clove oil is used as a fragrance for cosmetics and drugs. Antimicrobial, insect repellent, and hygiene have been reported and used as food, drug preservatives and dental painkillers, and are known to be safe and nontoxic (Bremness L, 1988, The complete book of herbs, Dorling Kindersley, London; Ok-Hwan Lee et al., Journal of the Korean Society of Food Science and Nutrition, 2004, 494-499). The components reported to date include beta-carotene, beta-pinene, beta-sitosterol, camphorsterol, carbon, cariophylene, chabicol, cinnamonaldehyde, ellagic acid, eugenol, gallic acid, camphorol, linarool, methyl Rugenol, musilage, olenolic acid, stigmasterol, tannin, vanillin and the like. Recently, anticoagulant active fractions have been reported from cloves (Lee Jong-im et al., Korean Society of Food Science and Nutrition, 2000, 712-718), but active substances have not been identified by ultrafiltration fractions of alkaline extracts (Lee Jong-im et al., Korean Food). Korean Journal of Nutrition Science, 2000, 543-548.

On the other hand, 2-alpha-hydroxy-oleanolic acid (2α-hydroxy-oleanolic acid) has not been reported anti-thrombotic activity, but strong antimicrobial activity against caries is reported (Public Patent No. 2002-0068424), Oleanoic acid, a dehydroxy form thereof, is extracted from Kochia scoporia and has been reported to have an anti-angiogenic activity (Patent Publication 199-0011004).

Accordingly, the present inventors aimed at developing functional foods exhibiting anti-thrombotic and blood flow improving activity, while searching for thrombin inhibitory activity from various natural products, purely separating powerful thrombin inhibitory substances from cloves, and identifying their active sites. The present invention was completed by confirming the stability under various conditions, for example, thermal, acid and plasma stability.

Accordingly, an object of the present invention is 2-alpha-hydroxy-oleanolic acid having thrombin inhibitory activity from various natural products such as cloves, jujube, bokbunja, olive tree, Chinese quince tree, loquat tree and Chinese hydrangea tree. I want to separate

The object of the present invention is to powder methanol extracts of various natural products, such as cloves, jujube, bokbunja, olive tree, Chinese quince tree, loquat tree, Chinese hydrangea, etc. under reduced pressure, and sequential to hexene, chloroform, ethyl acetate, butanol and water. The fractions were prepared, and the ethyl acetate fractions were subjected to continuous silica gel chromatography to isolate 2-alpha-hydroxy-oleanolic acid, which is a triterpenoid compound, and to determine the physicochemical properties of the material. This was accomplished by identifying the structures, investigating their stability and identifying the active sites of the material.

EMBODIMENT OF THE INVENTION Hereinafter, the structure of this invention is demonstrated concretely.

The present invention comprises the steps of preparing a clove extract having a thrombin inhibitory activity; Purifying the active substance from the extract; Elucidating the physicochemical properties and structure of the purified material; Investigating the stability of the material; And a step of identifying the active site of the substance.

2-alpha-hydroxy-oleanolic acid of the present invention is prepared by methanol extraction of clove dried flowers, sequential organic solvent fractions, and serial silica gel chromatography of ethyl acetate fractions in fractions.

Clove of the present invention is an important spice that has been used since ancient times regardless of East and West, and is registered as an edible raw material in Korea (Food and Drug Administration, 2003; www.kfda.go.kr ), thereby securing the safety of raw materials.

Clove extract of the present invention can be prepared using a variety of extraction solvents. The extraction solvent includes water, chloroform, alcohol, anhydrous or hydrous lower alcohol having 1 to 4 carbon atoms (methanol, ethanol, alcohol, propanol, butanol, etc.), and a mixed solvent of the lower alcohol and water. Preferably, extraction with methanol shows the maximum yield of active material (Example 1). Clove extract of the present invention can be prepared into a powder through a conventional powdering process, such as distillation and freeze-drying, or spray drying.

Clove extract prepared using the above extraction solvent, such as alcohol, water, methanol, ethanol, and then suspended in water, and then sequentially partitioned using hexene, chloroform, ethyl acetate and butanol, and divided into remaining water fractions. In this case, the active substance is detected in the chloroform, ethyl acetate and butanol fractions, and the ethyl acetate fraction is the most efficient (Example 2). The ethyl acetate fraction may be prepared as a powder through a conventional powdering process such as distillation and freeze drying or spray drying, and the active substance may be purified through silica gel chromatography (Example 2, FIG. One).

In addition, the present invention 2-alpha-hydroxy-oleanolic acid (2α-hydroxy-oleanolic acid) is not only a clove, but also a variety of natural products such as jujube, bokbunja, olive, Chinese quince, loquat, loquat, Chinese hydrangea, etc. It can be manufactured according to the present invention, and is not particularly limited only to cloves. In the following embodiment, the clove is described as an example.

EXAMPLE

Example 1 Extraction of Thrombin Inhibitor from Clove

Dried cloves ( S. aromaticum ) buds were purchased from the Korean medicine market (Andong, Gyeongbuk) and used as samples. Finely chopped clove (3,320g) was extracted for 6 hours using a reflux extractor at 65 ℃ using methanol, and this was repeated three times to recover the extract, concentrated under reduced pressure to obtain a methanol extract sample (187.2g). Thrombin inhibitory activity of the prepared methanol extracts was evaluated by measuring thrombin time (Sohn et al ., 2004. Kor. J. Pharmacogn 35. 52-61; Kwon et al. , 2004. J. Life Science , 14. 509 -513). Plasma was prepared from the volunteer's whole blood, and immediately after the blood was collected, centrifuged at 5,000 × g for 5 minutes at 4 ° C. to separate the plasma and stored frozen (fresh frozen plasma), and thawed at room temperature if necessary. The measurement method of thrombin time is as follows.

Thrombin time; 50 µl of 0.5 U thrombin (Sigma Co., USA), 50 µl of 20 mM CaCl ₂ , and 10 µl of various concentrations of the sample extract were mixed in a tube of Amelung coagulometer KC-1A (Japan) and reacted for 2 minutes. After addition of 100 μl, the time until the plasma was coagulated was measured. Aspirin (Sigma Co., USA) and heparin (heparin; Sigma Co., USA) were used as controls, and DMSO was used instead of the sample as a solvent control. DMSO showed a solidification time of 32.1 seconds. In the case of thermal stability measurement, sample solutions of various concentrations were heat-treated at 100 ° C. for 30 minutes, allowed to cool at room temperature for 1 hour, and then residual activity was measured. The thrombin inhibitory effect was expressed as the average value of the experiment repeated three or more times, and the solidification time at the time of sample addition divided by the solidification time of the solvent control was multiplied by 100 and expressed as%.

Aspirin used for the treatment of thrombosis showed a thrombin inhibition rate of 185 ± 13.2% at a concentration of 1 mg / mL, and methanol extract of clove showed a inhibition rate of 151 ± 25.6%, indicating a thrombin inhibitory effect of purified aspirin.

Example 2: Purification of Active Substance by Sequential Organic Solvent Fraction of Clove Methanol Extract and Silica Gel Chromatography

The methanol extract (187.2 g) of the clove prepared in Example 1 was sequentially partitioned in the order of hexene, chloroform, ethyl acetate, butanol, and the fractions and water residue were concentrated under reduced pressure to give 54.49 g of hexene fraction and 27.95 g of chloroform fraction. To give 50.0 g of ethyl acetate fraction, 26.64 g of butanol fraction and 28.12 g of water residue, respectively. Thrombin inhibitory activity of each fraction was 105 ± 3.5%, 143 ± 6.2%, 181 ± 21.8%, 281 ± 3.2%, 113 ± 4.2% at 1mg / mL concentration, respectively. In consideration of the ethyl acetate fraction was attempted to purely separate the active material.

50 g of ethyl acetate fraction was loaded on a column according to a conventional silica gel chromatography method, and then hexene-ethyl acetate (farm tool effluent), chloroform-methanol (10: 1 v / v) and chloroform-methanol (8: 1 v / v). ), Chloroform-methanol-water (8: 2: 0.5 v / v / v), Chloroform-methanol-water (7: 3: 1 v / v / v), Chloroform-methanol-water (52: 28: 8 v / v / v) and sequentially divided into 16 fractions, of which 9 and 10 fractions were collected and subjected to silica gel chromatography again, and the effluent solvent was chloroform-methanol-water (8: 2: 0.5). v / v / v) was used to finally isolate the active material. The thrombin inhibitory activity of the isolated active substances is shown in Table 1, wherein a control group, aspirin and heparin, which is used as an antithrombotic prophylactic and therapeutic agent, was used.

Thrombin Inhibitory Activity by Concentration of Substances Isolated from Clove Concentration (㎍ / mL) Thrombin inhibitory activity (%) Heparin Isolation aspirin 0 100.0 ± 1.1 100.0 ± 1.0 100.0 ± 1.1 1.0 125.4 ± 31.2 - - 1.5 > 2,000 - - 2.0 > 2,000 - - 15.6 - - - 31.3 - - - 62.5 - - - 125.0 - - 115.8 ± 5.5 375.0 - 139.5 ± 8.6 - 500.0 - > 2,000 136.7 ± 16.3 1000.0 - > 2,000 301.3 ± 30.2 1500.0 - > 2,000 290.5 ± 50.4

-: Unmeasured

As shown in Table 1, the isolate showed weak activity compared to heparin, but showed very strong thrombin inhibitory activity compared to aspirin.

Example 3: Physicochemical Properties and Structural Characterization of Isolated Active Substances

The physicochemical characteristics of the pure material isolated in Example 2 were analyzed and the structure was identified. Melting point was used with a Yanaco micro-melting point apparatus, not calibrated, RI spectra were used with Mattson Polaris TM (FT-IR) spectrometer, UV spectra were with Varian DMS 200 UV-Vis spectrophotometer It was. The EI-MS and FAB-MS spectral values were recorded with JMS SX-102A and JMS HX-110 / 110A (JEOL) spectrometers. ¹ H- and ¹³ C-NMR spectral values were recorded on TMS Bruker DRX-500 or Bruker AMX-300 spectrometer, expressed in ppm. Kieselgel 60 F ₂₅₄ plates (Merck, 5715) were used for TLC analysis. The analysis results were as follows, and ¹³ C-NMR analysis results are shown in Table 2.

¹³ C-NMR (pyridine- d ₅ ) Analysis of Isolated Active Compound Carbon number Isolation Carbon number Isolation C-1 46.7 C-16 23.9 C-2 68.9 C-17 47.8 C-3 83.8 C-18 42.0 C-4 39.8 C-19 46.4 C-5 55.9 C-20 31.0 C-6 18.9 C-21 34.2 C-7 33.3 C-22 33.3 C-8 39.8 C-23 29.3 C-9 48.2 C-24 17.5 C-10 38.6 C-25 16.9 C-11 23.8 C-26 17.7 C-12 122.5 C-27 26.2 C-13 144.9 C-28 180.2 C-14 42.2 C-29 33.3 C-15 28.3 C-30 23.7

The structure of the isolated active substance based on the above results is shown in FIG. 1, and identified as 2-alpha-hydroxy-oleanolic acid. In addition, the characteristics of the material are as follows:

Isolate: White needle crystal obtained from methanol,

mp 276-278, LB test: positive.

IR v _max 3418 (OH), 1698 (C = O), 1634 (C = C), 1049 cm ^-1 .

EI-MS m / z (rel. Int.) 427 [M] ⁺ (3), 428 [MCOO] ⁺ (2), 248 [D / E ring] ⁺ (28), 203 [D / E ring-COOH ] ⁺ (10), 189 (2) 55 (100).

¹ H-NMR (pyridine-d5) d: 5.46 (1H, brs, H-12), 4.07 (1H, d, J = 4.5, 9.0 and 11.2 Hz, H-2), 3.38 (1H, d, J = 9.3 Hz, H-3), 3.29 (1H, dd, J = 4.0 and 13.0 Hz, H-18)

Example 4 Stability of Thrombin Inhibitory Activity of Acid, Heat, and Plasma Treatment of 2-alpha-hydroxy-oleanolic Acid of the Present Invention

The thermal stability, acid stability and plasma stability of the isolated active substance were evaluated, and the activity was decreased even after 2 hours at 100 ° C, 2 hours at pH 2 (0.01M HCl) and 2 hours at plasma. It did not appear, indicating high stability. Plasma used in the experiment was used to adjust the final concentration of fresh frozen plasma and calf plasma (FBS: fetal bovine serum) to 50%. Aspirin used as a control was very unstable in saline solution (0.85% NaCl) and very unstable in plasma. In particular, aspirin showed 290.5 ± 50.4% thrombin inhibitory activity at a concentration of 1,5 mg / mL (Example 2, Table 1), but lost activity within 10 minutes, while heparin and 2-alpha-hydroxy- Oleanoic acid (2α-hydroxy-oleanolic acid) showed excellent stability in plasma and saline solution at more than 2,000% inhibition at the concentration of 1.5㎍ / mL and 0.5mg / mL, respectively.

Example 5 Active Site and Thrombin Inhibitory Effect of 2-alpha-hydroxy-oleanolic Acid of the Present Invention

Thrombin inhibitory activity against structurally similar substances was evaluated to investigate the active site and thrombin inhibitory mechanism of 2-alpha-hydroxy-oleanolic acid. As shown in Table 3, various triterpenoid compounds were hardly recognized for thrombin inhibitory activity, especially in the oleanolic acid which is a dehydroxy form of 2-alpha-hydroxy-oleanolic acid. Did not appear. These results indicate that 2α-hydroxy of 2-alpha-hydroxy-oleanolic acid plays an important role in inhibiting thrombin activity.

Thrombin Inhibitory Effect of Triterpenoid Compounds with Structures Similar to 2-alpha-hydroxy-oleanolic Acid Isolated material (triterpene system) Concentration (mg / mL) Thrombin inhibitory activity (%) 2-alpha-hydroxy-oleanolic acid 0.5 > 2,000 Oleanolic acid One 127.9 Oleanolic acid-3-o-glucuronic acid diester 0.5 111.8 Cysterone 0.5 117.1 Ecdysterone 0.5 109.2 Pistolside A 0.5 98.4 Pistolside B 0.5 98.2 Pistolside C 0.5 91.3 Ronnie Seroside A 0.5 108.2 Spicatoside A 0.5 101.6 Silcioside A 0.5 97.6 Inosterosterone 0.5 107.9 Euscapic acid 0.5 139.2

As can be seen from the above results, 2-alpha-hydroxy-oleanolic acid isolated from cloves shows strong inhibitory activity against human thrombin and also in heat treatment, acid treatment and plasma treatment. It was found that it can be used in various processing processes when used as a composition for preventing and treating thrombin-inhibited thrombosis due to its excellent stability.

Example 6: Preparation of 2-alpha-hydroxy-oleanolic acid from oleanolic acid

In Example 5, 2α-hydroxy of 2-alpha-hydroxy-oleanolic acid was found to be an active site that plays an important role in thrombin activity inhibition. In general, oleanolic acid is readily available, but 2-alpha-hydroxy-oleanolic acid could not be obtained without pure separation and purification in plants. Therefore, if a commercially available oleanoic acid is capable of mass production of 2-alpha-hydroxy-oleanolic acid via structural modification, 2-alpha-hydroxy-oleanolic acid is possible. The development and prevention of thrombin-inhibited thrombosis with) will give higher economics.

Accordingly, it was confirmed that 2-alpha-hydroxy-oleanolic acid having an active group of 2α-hydroxy was prepared by hydroxylation on the second carbon of oleanolic acid according to a conventional method.

As a result, 2-alpha-hydroxy-oleanolic acid having the structure of FIG. 1 was obtained.

The preparation of 2-alpha-hydroxy-oleanolic acid through hydroxylation of oleanoic acid would be very effective in terms of cost reduction.

As described through the above embodiment, the present invention relates to a composition for preventing and treating thrombin-inhibited thrombosis containing 2-alpha-hydroxy-oleanolic acid, cloves, jujube, bokbunja, olives 2-alpha-separated from various natural products such as trees, Chinese quince, loquat, loquat and Chinese hydrangea using water, spirits, methanol, ethanol, butanol, etc. There is an excellent effect of providing hydroxy-oleanolic acid (2α-hydroxy-oleanolic acid). In addition, the present invention 2-alpha-hydroxy-oleanolic acid (2α-hydroxy-oleanolic acid) exhibits excellent thrombin inhibitory activity, can effectively suppress the formation of thrombus, and through thrombosis, thrombosis such as ischemic stroke and hemorrhagic stroke It can be used for the prevention and treatment of In addition, it is isolated from the cloves and loquat leaves that have been used for food, and has excellent safety, and shows stable activity even during heat and acid treatment and plasma treatment. It can be prepared. Therefore, the present invention is a very useful invention in the pharmaceutical industry.

Claims (5)

A method for producing 2-alpha-hydroxy-oleanolic acid, characterized in that it comprises extracting the clove with water or an organic solvent, sequential organic solvent fraction and silica gel chromatography. The method of claim 1, wherein the water or organic solvent is selected from water (cold water or hot water), spirits, anhydrous or hydrous alcohols having 1 to 4 carbon atoms, or 2-alpha- characterized in that the mixed solvent of the alcohol and water. Method for preparing hydroxy-oleanolic acid (2α-hydroxy-oleanolic acid). A composition for preventing and treating thrombin-inhibited thrombosis, comprising 2-alpha-hydroxy-oleanolic acid prepared according to the method of claim 1 as an active ingredient. Anticoagulant, blood flow preventing and treating composition, characterized in that it comprises water or organic solvent extract of the clove, fractions of the extract or modulator of silica gel chromatography, and isolating material and a pharmaceutically acceptable carrier. delete

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