KR20040008014A - Extracts of piper longum having potent inhibitory effect on platelet aggregation and uses thereof - Google Patents

Abstract

PURPOSE: Provided is an extract of Piper Longum which exerts excellent platelet aggregation inhibiting effect, and is effectively used to prevent and treat cerebrovascular and cardiovascular diseases. CONSTITUTION: An extract of Piper Longum is obtained by extracting the root of Piper Longum with water or an organic solvent. The organic solvent is selected from the group consisting of alcohol, hexane, chloroform, ethyl acetate, and butanol. The extract comprises alkaloid-based compounds selected from the group consisting of piperlongumine, pipernonaline, piperettine, pipereoctadecalidine, and derivatives thereof.

Description

Extract of essential tree with excellent platelet aggregation inhibitory activity and use thereof {EXTRACTS OF PIPER LONGUM HAVING POTENT INHIBITORY EFFECT ON PLATELET AGGREGATION AND USES THEREOF}

[Industrial use]

The present invention relates to a piper longum extract having excellent platelet aggregation inhibitory activity and a use thereof, and more particularly, to a seedling tree extract useful for preventing and treating brain and cardiovascular diseases due to its excellent platelet aggregation inhibitory activity. will be.

[Private Technology]

In general, diseases are expressed in various forms as social, economic, cultural and environmental factors change. Recently, the rapid economic development and the improvement of living standards have led to the diversification of lifestyles, including the diet, which has led to the reduction of past infection-oriented diseases and the increase of advanced degenerative diseases.

Korea's death toll in 1993 shows 523.8 deaths per 100,000 population, among which 37.3% of circulatory diseases, including 27.3 hypertensive diseases, 43.3 heart diseases, and 83.8 cerebrovascular diseases, account for 30.2% of the total. Is increasing. As a result, the mortality rate of cerebral and cardiovascular diseases such as hypertension, arteriosclerosis, ischemic heart disease, cerebral infarction, cerebral hemorrhage and stroke has increased about 6 times in the past decade.

Brain and cardiovascular diseases are closely related to blood as the main cause of the increase of blood cholesterol, the change of blood components according to the change of lipid composition and excessive mental tension. Under normal conditions, blood does not coagulate in blood vessels, but when bleeding occurs, one of the body's defenses to blood clotting occurs. However, a blood clot (thrombus), which is created when blood vessels are damaged due to changes in blood components or when the internal diameter of the blood vessels is narrowed due to arteriosclerosis, prevents the coronary arteries or microcerebrovascular vessels that supply blood to the heart through the blood flow. Brain and cardiovascular disease develops. Also, due to the progress of the disease, the risk of recurrence is high, so most cases require lifelong treatment.

Platelets play an important role in thrombosis and haemostasis, which cause circulatory disorders. When blood vessels are damaged by various causes, platelets aggregate and irreversibly adhere to the deformed surface. A thrombus is a blood-driven mass in the blood vessels that is damaged by pathological conditions, trauma, or other chemicals, especially when collagen is exposed to collagen, causing platelets to adhere to it. . Adhesed platelets release ADP, thrombin, calcium, and the like, form thromboxin A ₂ (TXA ₂ ) to activate and aggregate peripheral platelets. These aggregates fall off by the bloodstream and are called thrombus, and the thrombus flows along the bloodstream and blocks microvascular blood vessels such as myocardial infarction and ischemia.

Recently, studies on natural products having excellent antiplatelet aggregation and anticoagulant activity have been actively conducted for the prevention and treatment of such chronic degenerative diseases, particularly circulatory diseases. In particular, bioactive functions such as maintaining biological defense and homeostasis by food ingredients, preventing disease and fast healing are recognized as a good coping method for diseases related to diet, and are being actively researched and developed.

For example, ajoene, which has a platelet aggregation inhibitory activity, has been isolated from green onions ( Allium fistulosum ), garlic ( Allium sativum ) and onions ( Allium cepa ) which have been used for diseases such as blood and blood. A new antiplatelet agent is being developed that blocks the transformation to fibrin. In addition, curcumin (diferuloylmethane), the main ingredient of turmeric, a spice widely used in the Orient, has a strong inhibitory effect with an IC ₅₀ value of 20-25 μM for PAF and arachidonic acid. Is known to be a platelet aggregation inhibitor, and its mechanism of action is known to inhibit TXA ₂ synthesis and Ca ²⁺ signaling ( Biochem. Pharmacol ., 58, 1167-1172, 1999).

In addition, domestic and international researches on the search for physiologically active substances derived from environmentally friendly natural products have received many achievements, but there is still insufficient research.

In order to solve the above problems, an object of the present invention is to provide an essential tree extract excellent in platelet aggregation inhibitory activity.

Another object of the present invention is to provide an alkaloid compound isolated from the extract of the essential tree.

Still another object of the present invention is to provide a pharmaceutical composition for treating and preventing brain and cardiovascular diseases, including an essential tree extract or an alkaloid compound isolated from the extract.

Still another object of the present invention is to provide a food for preventing brain and cardiovascular diseases, including an essential tree extract or an alkaloid compound isolated from the extract.

In order to achieve the above object, the present invention provides a seedling tree extract excellent in platelet aggregation inhibitory activity obtained by extracting the root of the seedling tree ( Piper Longum ) with an extraction solvent.

The present invention is also selected from the group consisting of piperlongumine, pipernonaline, piperettine, pipereoctadecalidine and derivatives thereof, isolated from the essential tree extract. It provides an alkaloid compound.

The present invention also provides a pharmaceutical composition for the treatment and prevention of brain and cardiovascular diseases, including the essential tree extract or an alkaloid compound isolated from the extract.

The present invention also provides a food for the prevention of brain and cardiovascular diseases, including the above essential tree extract or an alkaloid compound isolated from the extract.

Hereinafter, the present invention will be described in detail.

Extracts excellent in the platelet aggregation inhibitory activity of the present invention are obtained from the essential tree roots. Trees have been used for thousands of years in India to relieve cultivation, walls, wags, chills, colds, heartaches and blood ( Indian Drugs, June, 384-388, 1984 ). It is used as a herbal medicine under the name of (必 發). Significant studies have been conducted on the activity of the essential tree, and medically anti-allergic effects ( Indian J. Physiol. Pharmacol ., 43: 486-490, 1999 ), anti-inflammatory effects ( Indian J. Exp. Biol., 32: 633 -636, 1994 ) and hepatoprotective effects ( Planta Med., 59: 413-417, 1993 ).

The essential tree extract of the present invention is prepared by extracting the essential tree root with an organic solvent. Water or an organic solvent can be used as an extraction solvent, It is preferable to use an organic solvent. According to a preferred embodiment of the present invention, the extract of crude hexane, chloroform, ethyl acetate, butanol and water is prepared by sequential solvent fractionation after crude extraction of the tree. These extracts obtained from the roots of the essential tree have excellent platelet aggregation inhibitory activity. Among them, the chloroform fraction of the essential tree is preferred because it contains the most compound with excellent platelet aggregation inhibitory activity.

The present invention provides an alkaloid compound obtained by separating and purifying the essential tree extract by silica gel chromatography and high performance liquid chromatography (HPLC). The alkaloid compound was separated from the crude extract of the tree by sequential solvent fractionation, and then hexane, chloroform, ethyl acetate, butanol and water fractions were separated from the chloroform fractions by silica gel chromatography and high-performance liquid chromatography. It can be obtained by purification. Such isolated and purified alkaloid compounds include piperlongumine ( N- (3,4,5-trimethoxycinnamoyl) -Δ ³ -pyridin-2-one) and pipernonaline; (2 E, 8 E) - N - [9- (3,4- methylenedioxyphenyl) Noil the non-Nadi 2,8] piperidine), piperazine retinoic (piperettine; (2 E, 4 E, 6 E ) - N - [7- (3,4- methylenedioxyphenyl) heptyl-2,4,6-tart Rie Russo] piperidine), blood Ferre octadecyl potassium Dean (pipereoctadecalidine; (2 E, 4 E, 12 Z) - N -. (Russo in 2,4,12- octahydro big tree)] piperidine, and the like of these substituted derivatives is the most preferable in the blood Peron constituency double.

The essential tree extract of the present invention and the alkaloid compound isolated and purified from the extract have excellent platelet aggregation inhibitory activity and thrombus formation inhibitory activity, and thus can be used as antiplatelet aggregation agent and thrombosis agent.

Collagen, PAF and arachidonic acid are known as platelet inducers that primarily activate platelets by the cyclooxygenase pathway. The essential tree extract of the present invention and the alkaloid compound isolated and purified from the extract have excellent platelet aggregation inhibitory activity against collagen, PAF, and arachidonic acid, which are factors for platelet activation.

The essential tree extract, alkaloid compounds isolated and purified from the extract, substituted derivatives thereof, or pharmaceutically acceptable salts thereof have excellent anti-platelet aggregation or thrombosis due to their excellent platelet aggregation inhibitory activity and thrombus formation inhibitory activity. It can be used as a therapeutic agent for preventing and treating the same brain and cardiovascular diseases. The brain and cardiovascular diseases include high blood pressure, heart disease, cerebrovascular disease, arteriosclerosis, ischemic heart disease, cerebral infarction, cerebral hemorrhage, stroke, and the like.

Suitable formulations of the therapeutic agents include, but are not limited to, tablets, dragees, hard or soft capsules, solutions, suspensions or emulsions, injections, suppositories, and the like. The extract or alkaloid compound, substituted derivatives thereof or salts thereof may be prepared in a suitable formulation using a pharmaceutically inert organic or inorganic carrier. That is, when the formulation is a tablet, coated tablets, dragees and hard capsules, lactose, corn starch or derivatives thereof, talc, stearic acid or salts thereof can be used. Also in the case of soft capsule formulations, polyols of vegetable oils, waxes, fats, semisolids and liquids can be used. In the case of solution or syrup form, water, polyols, glycerol, vegetable oils and the like can be used. As suppository carriers, natural or hardened oils, waxes, fats, liquid polyols and the like can be used.

The therapeutic agent of the present invention may further include preservatives, stabilizers, wetting agents, emulsifiers, solubilizers, sweeteners, colorants, osmotic pressure regulators, antioxidants and the like.

The method of administration can be readily selected according to the dosage form and can be administered orally or parenterally. The dosage may vary depending on the weight of the patient, the severity of the condition, sex, and age. However, in the case of a healthy adult male, the dosage is preferably 0.1 to 50 mg / kg per day. The number of doses can also be easily adjusted according to the weight, severity, sex, and age of the patient.

Also added to various foods can be used in functional foods for preventing brain and cardiovascular diseases. The tree extract of the present invention may be added in a liquid state or may be lyophilized and powdered. The food to be added may be any food in the market and is not particularly limited.

EXAMPLE

Hereinafter, a preferred embodiment of the present invention. However, the following examples are only presented to aid the understanding of the present invention, and the present invention is not limited to the following examples.

Example 1 Isolation and Structure Verification of Alkaloid Compounds

Primary tree roots were sold by the US Department of Agriculture's West Regional Research Center (WRRC) and dried in the shade. 10.0 kg of the dead tree roots were ground, put into 20 L of methanol, and extracted for 6 days while shaking with a shaker. After 6 days, the filtrate was concentrated under reduced pressure at a temperature of 30 ° C. or lower to obtain a crude methanol extract. This process was repeated twice to obtain 3.0 kg of crude methanol extract.

20 g of this crude methanol extract was mixed with 800 ml of distilled water, poured into a 2000 ml fraction funnel, and then mixed with hexane in the same amount. The mixture was shaken to separate the water and hexane layers, and the process was repeated. Got it. The remaining water layer was repeatedly extracted with 800 ml of chloroform twice to give 1600 ml of chloroform layer. The remaining water layer was repeatedly extracted twice with 800 ml of ethyl acetate to obtain 1600 ml of ethyl acetate layer and water layer, and repeatedly extracted twice with 800 ml of butanol to obtain butanol layer and water layer. The hexane layer, the chloroform layer, the ethyl acetate layer, and the butanol layer were concentrated under reduced pressure, and then separated by silica gel chromatography containing silica gel having a particle size of 25-100 μm. The separated fractions were separated by high performance liquid chromatography (HPLC) to obtain an alkaloid compound. The structure of the separated alkaloid compound was confirmed by ¹ H-NMR and ¹³ C-NMR. The solvent for HPLC analysis was TEDIA, and CDCl ₃ -d ₃ (0.03% TMS) (manufactured by Aldrich) was used as the solvent for NMR analysis.

Alkaloid compounds isolated and purified by the above method include piperlongumine ( N- (3,4,5-trimethoxycinnamoyl) -Δ ³ -pyridin-2-one) and pipenoline (pipernonaline; (2 E, 8 E) - N - [9- (3,4- methylenedioxyphenyl) Noil the non-Nadi 2,8] piperidine), piperazine retinoic (piperettine; (2 E, 4 E, 6 E) - N - [7- (3,4- methylenedioxyphenyl) heptyl-2,4,6-tart Rie Russo] piperidine), blood Ferre octadecyl potassium Dean (pipereoctadecalidine; (2 E, 4 E, 12 Z) - N - ( Russo in 2,4,12- octahydro big tree) blood were identified, such as piperidine, and their substituted derivatives.

Example 2: Platelet Aggregation Inhibitory Activity

The rabbit arteries (weight: 2.5-3.5 kg) were expanded with 70% ethanol, and 30 ml of blood was collected using a 20G needle, and a citrate-dextrose solution (65 mM cirtric acid, 85 mM trisodium citrate, 2% dextrose, pH 4.5) was suspended in 5 ml, and then centrifuged at 1,600 rpm for 10 minutes. The supernatant was separated, centrifuged at 1,400 rpm for 5 minutes to remove precipitates, and centrifuged again at 2,600 rpm for 10 minutes to remove the supernatant. The precipitate was washed twice with Tyrode HEPES buffer (pH 6.35) solution, centrifuged at 2,500 rpm for 10 minutes and finally suspended with Tyrode HEPES buffer (pH 7.35) solution to prepare washed platelets. Platelet counts were measured with an optical microscope and a hemocytometer to dilute with Tyrode HEPES buffer (pH 7.35) solution to 4 × 10 ⁸ / ml and used for the experiment. The composition of Tyrode HEPES buffer is shown in Table 1.

Composition of Tyrode HEPES Buffer (pH 7.35) Ingredient density NaClKClMgCl ₂ 6H ₂ ONaCO ₃ HEPES albumin (Bovine) glucose 138.3 mM 2.68 mM 1.048 mM 4.0 mM 10 mM 0.35% 0.1%

250 μl of platelet suspension is taken in the cuvette and the CaCl ₂ is diluted to a final concentration of 1 mM. After incubation at 37 ° C. for 3 minutes with stirring at 1,200 rpm, each sample (alkaloid compound) was injected to a final concentration of 1 mg / ml, and then incubated for 3 minutes, and then platelet inducers were administered at an optimal concentration. Optimal concentrations of platelet triggers were as follows: collagen: 2 μg / ml, PAF: 10 nM, arachidonic acid: 100 μM.

The aggregation rate of platelets by each platelet inducing factor was evaluated by the light transmittance change measured using an absorbance measuring device. All experiments were statistically repeated three times.

Platelet activation was expressed as percent aggregation and the results for platelet aggregation were expressed as percent inhibition relative to the control group, and the control group was measured using DMSO instead of the sample and is shown in Table 2. Inhibition rate (%) was calculated according to the following equation (1).

[Equation 1]

% Inhibition = [(A-B) / A] × 100

A is the aggregation rate of the control and B is the aggregation rate of the sample.

extract Concentration (µg / ml) Collagen (2 μg / ml) PAF (10 nM) Arachidonic acid (100 μM) Cohesion Rate (%) % Inhibition Cohesion Rate (%) % Inhibition Cohesion Rate (%) % Inhibition Control 72.7 ± 2.9 - 69.3 ± 1.8 - 69 ± 2.0 - Hexane layer 100 7 ± 0.3 ^** 96 5 ± 0.1 ^** 93 9 ± 0.1 ^** 87 50 39 ± 1.1 ^* 46 36 ± 0.5 ^** 48 22 ± 0.7 ^* 68 Chloroform layer 100 3 ± 0.3 ^** 96 3 ± 0.2 ^** 96 1 ± 0.3 ^** 99 50 12 ± 0.6 ^** 84 10 ± 0.7 ^** 86 8 ± 1.5 ^** 88 10 23 ± 3.1 ^** 68 27 ± 1.4 ^** 61 17 ± 1.8 ^** 75 5 35 ± 2.1 ^* 52 34 ± 1.8 ^** 51 33 ± 1.8 ^** 52 Ethyl acetate layer 100 10 ± 0.5 ^** 86 5 ± 0.4 ^** 93 8 ± 0.8 ^** 88 50 23 ± 1.3 ^* 68 23 ± 0.9 ^** 67 29 ± 1.4 ^** 58 10 45 ± 2.1 ^* 38 55 ± 1.8 ^* 20 50 ± 2.3 ^* 28 Butanol layer 100 24 ± 1.1 ^** 67 11 ± 0.4 ^** 84 19 ± 0.4 ^** 72 50 57 ± 1.8 ^* 22 39 ± 1.8 ^** 43 37 ± 1.3 ^** 46 Water layer 100 35 ± 3.2 ^* 52 29 ± 1.5 ^** 58 20 ± 1.0 ^** 71 50 47 ± 1.0 ^* 36 44 ± 2.1 ^* 36 55 ± 1.3 ^** 20

Note) Cohesion rate is mean ± standard deviation (n = 3)

* P <0.05, ** P <0.01

As shown in Table 2, when 2 μg / ml of collagen was administered as a platelet inducer, 96, 96, 86, 67, and 52% of hexane, chloroform, ethyl acetate, butanol and water extracts at 100 μg / ml, respectively, were used. When 10 nM of PAF was administered, 100 ㎍ / ml of hexane, chloroform, ethyl acetate, butanol and water extracts showed 93, 96, 93, 84 and 58% of inhibitory effects, respectively. When administered at 100 μM of arachidonic acid, hexane, chloroform, ethyl acetate, butanol and water extracts showed inhibitory effects of 87, 99, 88, 72 and 71%, respectively, at 100 μg / ml.

IC ₅₀ was calculated from the platelet aggregation inhibition rate and the sample concentration of hexane, chloroform, ethyl acetate, butanol and water extract, and is shown in Table 3 below.

extract IC ₅₀ (μM) Collagen (2 μg / ml) PAF (10 nM) Arachidonic acid (100 μM) Hexane layer 163 223 138 Chloroform layer 12.3 5.8 22.8 Ethyl acetate layer 152 278 181 Butanol layer 190 191 168 Water layer 255 237 175

As shown in Table 3, the hexane, chloroform, ethyl acetate, butanol and water extracts of the present invention had IC ₅₀ values of 12.3-255 μM, 5.8-237 μM, and 22.8-175 μM for collagen, PAF and arachidonic acid, respectively. Seemed. In the case of the chloroform extract, the IC ₅₀ value was 12.3, 5.8, and 22.8 μM against the platelet-inducing factor collagen PAF and arachidonic acid.

Example 3: Antithrombotic Effect of Chloroform Extract

Animal antithrombotic effect was verified by mouse thromboembolism test, referring to DiMinno and Silver method. Male ICR mice weighing 25-30 g that were stopped feeding for the night were used as experimental animals. As a sample, the chloroform extract obtained in Example 1 having the best platelet aggregation inhibitory effect was concentrated under reduced pressure, and dried powder was used.

After 90 minutes of oral administration of chloroform extract powder (50 mg / kg) suspended in a 0.5% aqueous solution of carboxymethyl cellulose (CMC), 114 μg of collagen and 13.2 μg of thromboembolism showing about 90% of thromboembolism Aqueous epinephrine solution was injected into the tail vein of the mice. The group administered orally with aspirin (50 mg / kg) was used as the positive control group, and the group administered orally with 0.5% carboxymethylcellulose aqueous solution was used as the negative control group. The number of dead or paralyzed mice was measured for 15 minutes and the percent (%) antithrombotic effect was calculated using Equation 2 below.

[Equation 2]

% Antithrombotic effect = [1-(death or paralyzed population) / total] × 100

Dose (mg / kg body weight) Death / Number of Test Subjects % Antithrombotic effect Negative Control ¹⁾ - 41/49 16.33 Chloroform extract 50 25/48 47.92 Positive control group ²⁾ 50 28/46 39.93

1) Negative control group: group administered orally with 0.5% carboxymethyl cellulose solution only

2) Positive control group: A group administered orally by suspending aspirin (50 mg / kg) in 0.5% carboxymethyl cellulose solution.

As shown in Table 4, oral administration of chloroform extract to mice showed a significant protective effect against death from pulmonary thrombosis. Chloroform extract of 50 mg / kg showed a protective effect of 47.92%, while aspirin (50 mg / kg) showed a 39.93% protective effect.

The root extract of the essential tree of the present invention and the alkaloid compound obtained therefrom are excellent in inhibiting the aggregation of platelets and can be usefully used for the prevention and treatment of brain and cardiovascular diseases.

All simple modifications or changes of the present invention can be easily carried out by those skilled in the art, and all such modifications or changes can be seen to be included in the scope of the present invention.

Claims (8)

A tree extract having excellent platelet aggregation inhibitory activity obtained by extracting the root of Piper Longum with water or an organic solvent. The method of claim 1, wherein the organic solvent is an extract of the tree is selected from the group consisting of alcohol, hexane, chloroform, ethyl acetate, and butanol. According to claim 1, wherein the extract is piperlongumine (piperlongumine), pipernonaline (pipernonaline), piperettine (piperettine), pipereoctadecalidine and alkaloids selected from the group consisting of derivatives thereof A tree extract containing the compound. Platelet aggregation inhibitory activity selected from the group consisting of piperlongumine, pipernonaline, piperettine, pipereoctadecalidine, and derivatives thereof, isolated from the roots of essential tree This excellent alkaloid compound. A pharmaceutical composition for treating and preventing brain and cardiovascular diseases, comprising the extract of the tree of claim 1. A pharmaceutical composition for treating and preventing brain and cardiovascular diseases comprising the alkaloid compound of claim 4. A food for preventing brain and cardiovascular diseases, comprising the extract of the tree of claim 1. A food for preventing brain and cardiovascular diseases, comprising the alkaloid compound of claim 4.