KR100728704B1 - Composition containing extract of stem bark of Rhus verniciflua or active single compounds isolated therefrom for Anti-thrombus - Google Patents

Abstract

The present invention relates to an anti-thrombotic composition comprising lacquer bark extract or an effective single substance isolated therefrom.

The lacquer bark extract according to the present invention is excellent in inhibiting platelet aggregation of rats induced by collagen in vitro, as well as inhibiting whole blood platelet aggregation in humans induced by various coagulation inducers, inhibiting platelet receptor expression and calcium in platelets. As well as an anabolic inhibitory effect and an excellent antithrombotic effect in vivo, it can be usefully used for the prevention and treatment of diseases involving thrombosis such as atherosclerosis, cerebral hemorrhage, stroke and cerebral infarction.

Description

Composition containing only anti-thrombotic bark extract or effective single substance isolated therefrom {Composition containing extract of stem bark of Rhus verniciflua or active single compounds isolated therefrom for Anti-thrombus}

1 is a view showing a process of extracting, separating from the bark bark according to the present invention.

The present invention relates to an anti-thrombotic composition comprising lacquer bark extract or an effective single substance isolated therefrom.

Cerebrovascular diseases such as atherosclerosis, cerebral hemorrhage, stroke and cerebral infarction and circulatory diseases such as heart disease are adult diseases that occupy the 1st and 2nd causes of death. As the adult disease develops into a modern society, it is frequently occurring in the middle-aged people due to changes in dietary habits and internal and external environmental stresses. The main cause of these diseases is known as thrombus [Ruggeri, ZM, Platelets in athrothrombosis, Nature Medicine 8, 1227-1234, 2002]. Thrombus is recognized as a pathology mediated by excess platelet aggregation. .

A thrombus is an aggregate of blood factors, mainly consisting of aggregates of platelets and fibrin or platelet aggregates, which are typically formed to prevent excessive bleeding of damaged blood vessels. When blood vessels are damaged, platelets are activated by stimulation of various aggregation inducers such as collagen, ADP or arachidonic acid, causing adhesion, secretion and aggregation, in which the platelet receptor P- Selectin, GPIIb / IIIa, and pituitary adenylate cyclase-activating polypeptide (PACAP) receptors act. These reactions are related to haemostasis and play an important role in the development of circulatory diseases, including thrombosis and the like.

Hemostasis is a mechanism to prevent bleeding from blood vessels whose original shape has been destroyed, through the process of activation of platelets, adhesion of platelets and vascular components, and the complex interactions of the associated coagulation factors. Removed through fibrin degradation [Harlan, JM and Harker, LA Haemostasis, thrombosis and thrombombolic disorders. Med . Clin . North. Am. , 65, 855, 1981.].

Platelets have a characteristic discoid form at rest, but when activated, they lose their inherent shape and undergo an irregular shape with fictitious shapes. Phosphorylation and subsequent interactions [Nachmias, VT, Kavaler, J. and Jacubowits, S. Reversible association of myosin with the platelet cytoskeleton. Nature , 313, 70, 1985.].

The antiplatelet agent developed so far and the like, theophylline (theopylline), mole sidomin (molsidomine), verapamil, and nifedipine, which is known for promoting the formation of cAMP and cGMP inhibit the mobilization of Ca ^{+ 2.} In addition, nonsteroidal compounds such as aspirin, imidazole and indomethacin are known as drugs having antiplatelet action by inhibiting the production of thromboxane A ₂ . However, although the drugs are chemicals as drugs, there are problems that cause various side effects such as excessive hemostasis, infertility, and digestive problems in the human body.

Therefore, a lot of research on the anti-thrombotic substances from natural products with few side effects has been conducted.

Meanwhile, Rhus verniciflua belongs to the family Anacardiaceae and is a deciduous broad-leaved arboreous tree that grows a lot in Northeast Asia such as China and Japan. The sap is called lacquer or dried lacquer, which is used for industrial and medicinal purposes. Especially, it is used as a medicine for poisoning, fever, schooling, insect repellent, abdominal pain, tuberculosis, constipation, and used as traditional food like lacquer chicken. Some studies on the physiological activity of the lacquer tree extract have been reported, and antimicrobial activity against the substances isolated from the lacquer tree, antioxidant activity against the flavonoid component and the urushiol component of sap, and anticancer activity have been reported. However, some of the components of the sumac have been reported as causing the allergic dermatitis, there is a problem as a drug and food development.

As such, many studies have been conducted on the sap of the lacquer tree, and there are few studies on the bark of the lacquer tree, which is known to have no side effects compared to the sap.

Therefore, the inventors of the present invention, while studying the anti-thrombotic substances in herbal medicine, the extract from the bark bark extract, fractions thereof and the effective single substance separated therefrom inhibits platelet aggregation, which has antithrombotic action, resulting in thrombosis formation It was confirmed that it can be used for the prevention and treatment of, and completed the present invention.

The present invention is to provide an anti-thrombotic composition comprising the sumac bark extract or an effective single substance isolated therefrom.

In addition, the present invention is to provide a platelet aggregation inhibitor comprising a sumac bark extract or an effective single material isolated therefrom.

The present invention provides an anti-thrombotic composition comprising a sumac bark extract or an effective single substance isolated therefrom.

In addition, the present invention provides a platelet aggregation inhibitor comprising the sumac bark extract or an effective single substance isolated therefrom.

Hereinafter, the present invention will be described in detail.

Extraction and separation of the lacquer bark extract according to the present invention is as follows.

The dried lacquer bark is pulverized, placed in a round flask and extracted with water, C ₁ -C ₄ alcohol, ethyl acetate or a mixture thereof, preferably 70% methanol, for 4-6 hours in a 70-90 ° C. water bath. . The above extraction process is repeated twice. The extract was left at room temperature, filtered through a filter paper, and concentrated under reduced pressure with an evaporator on a 55-65 ° C. water bath. The concentrate is suspended in distilled water, shaken by adding the same amount of ethyl acetate, and fractionated three times to separate the ethyl acetate layer and the water layer.

The platelet aggregation inhibitory effect of the ethyl acetate layer was confirmed to be excellent (Table 1), and the ethyl acetate layer was separated into five fractions (fractions 1 to 5) using column chromatography, preferably ODS column chromatography. Of these, fraction 2, the active fraction, is separated into four fractions (fractions 2-1 to 2-4) using column chromatography, preferably Sephadex LH-20 column chromatography. At this time, it is preferable to use 50% methanol as the solvent, it is preferable to use methanol as the mobile phase, more preferably 50% to 70% methanol is used.

Among the four fractions, isomaltol, which is one of the effective monoliths, was separated from fraction 2-2 by using preparative HPLC from the fraction 2-2 having excellent inhibition of collagen-induced whole blood platelet aggregation, and column chromatography was performed from fraction 2-3. To two fractions (fraction 2-3-1 and fraction 2-3-2). In addition, pentagalloyl glucose, which is an effective single substance, is separated from the fraction 2-3-2. Methanol is used as the solvent, and methanol, preferably 40 to 75% methanol, is used as the mobile phase.

Succulent bark extract according to the present invention, fractions thereof, and isomaltol and pentagalloylglucose, which are effective single substances isolated therefrom, are derived from collagen, adenosine 5 'phosphoric acid (ADP) or arachidonic acid in vitro. It is excellent in the inhibition rate of whole blood platelet aggregation of human.

In addition, isomaltol and pentagalloyl glucose according to the present invention inhibit the expression of platelet receptors GPIIb / IIIa, PAC-1 (type 1 PACAP receptor) and P-selectin, which are markers related to platelet activity. In addition, it significantly suppresses calcium mobilization in platelets. Therefore, since the platelet aggregation inhibition rate of the effective single substances according to the present invention is excellent, it can be usefully used in the composition for the prevention and treatment of diseases involving thrombus formation due to platelet activity such as atherosclerosis, cerebral hemorrhage, stroke and cerebral infarction.

The composition of the present invention may contain one or more active ingredients exhibiting the same or similar functions in addition to the lacquer bark extract, fractions thereof, or effective single substances separated therefrom.

In addition, the compositions of the present invention can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy and biological response modifiers for the prevention and treatment of diseases involving thrombus formation.

The composition of the present invention may be prepared by including one or more pharmaceutically acceptable carriers in addition to the above-described active ingredients for administration. Pharmaceutically acceptable carriers may be used in combination with saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol or one or more of these components, as necessary, and include antioxidants and buffers. And other conventional additives such as bacteriostatic agents can be added. In addition, diluents, dispersants, surfactants, binders or lubricants may be additionally added to formulate injectable formulations, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like. Furthermore, it may be preferably formulated according to each disease or component by a suitable method in the art or using a method disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA, 18th, 1990).

The composition of the present invention can be administered orally or parenterally (eg, applied intravenously, subcutaneously, intraperitoneally or topically) according to the desired method, and the dosage is based on the weight, age, sex and health of the patient. The range varies depending on the diet, the time of administration, the method of administration, the rate of excretion and the severity of the disease. In the case of the lacquer bark extract of the present invention or fractions thereof, the daily dose is 50 to 300 mg / kg, preferably 80 to 120 mg / kg, and in the case of an effective single substance separated therefrom, the daily dose is 50. It is -300 mg / kg, Preferably it is 100-150 mg / kg, It is more preferable to divide and administer once or several times a day.

Hereinafter, preferred examples and experimental examples are presented to help understand the present invention. However, the following Examples and Experimental Examples are provided only to more easily understand the present invention, and the contents of the present invention are not limited thereto.

Example  1: Preparation of Sumac extract and Separation of Effective Single Substance

The lacquer bark (standard number KIOM-01-02) used in this experiment was purchased from Jeongdo C & T (Seoul, Korea) and used for the experiment after confirming the external form in the inspection department of the Korea Institute of Oriental Medicine.

1-1. Extract from Sumac Bark

1.2 kg of dried lacquer bark was pulverized, placed in a round flask, and 6 L of 70% methanol was added and extracted for 5 hours on an 80 ° C water bath. The same extraction procedure was repeated twice. The extract was left at room temperature, filtered through a filter paper, and concentrated under reduced pressure with an evaporator on a 60 ° C. water bath.

The concentrated solution (423 g) was suspended in distilled water, shaken by adding the same amount of ethyl acetate, and fractionated three times, and separated into an ethyl acetate layer and a water layer.

As a result of the platelet aggregation inhibition test by the method of Experimental Example 1, it showed an excellent platelet aggregation inhibitory effect in the ethyl acetate layer among the extracts.

1-2. Isolation of Ethyl Acetate Extract

The ethyl acetate extract (59.4 g) obtained in Experimental Example 1-1 was separated into five fractions (fractions 1 to 5) using ODS column chromatography (Lichroprep RP-18, Merck) using 50% methanol as a mobile phase. . As a result of measuring the platelet aggregation inhibitory activity of these fractions by the method of Experimental Example 1, fraction 2 was the best (32.4 g).

The active fraction (fraction 2) was separated into four fractions (fractions 2-1 to 2-4) using Sephadex LH-20 column chromatography (Pharmacia) with 50% methanol as the mobile phase. As a result of measuring the platelet aggregation inhibitory activity of these fractions by the method of Experimental Example 1, fraction 2-2 (1.2 g) and fraction 2-3 (2.8 g) were excellent in platelet aggregation inhibitory activity.

1-3. Effective single substance  detach

From the fraction 2-2 (1.2 g) obtained in Example 1-2, isomaltol was separated by high performance liquid chromatography HPLC using 60% methanol as a mobile phase, and fraction 2-3 (2.8 g) was 45 Separation into two fractions (fractions 2-3-1 and 2-3-2) using an ODS column (Merck) chromatography with% methanol mobile phase. Among them, fraction 2-3-2 was separated from 70% methanol as a mobile phase by using Sephadex LH-20 column chromatography (Pharmacia) and silica gel column (Merck).

< Isomaltol>

Brown (10 mg), crystals from water or ether,

Mp 98 ~ 103 ℃,

C ₆ H ₆ O ₃ ,

R _f = 0.60 (RP)

FAB (-) Mass (m / z): 125 [MH] ^-

¹ H-NMR (600 MHz, CD ₃ OD): 7.88 (1H, d, J = 1.8 Hz, H-5), 6.84 (1H, d, J = 8.7 Hz, H-4), 2.52 (3H, s , H-1'methyl);

¹³ C-NMR (150 MHz, CD ₃ OD): 199.7 (C-3), 164.3 (C-5), 132.3 (C-2), 130.2 (C-1), 116.4 (C-4), 26.4 ( C-1′methyl).

< Pentagaloyl Glucose >

White powder (190 mg),

[D] ( c 0.76, MeOD),

C ₄₁ H ₃₂ O ₂₆ ,

ESI-MS m / z 941 [M] ⁺

¹ H-NMR (300 MHz, MeOD): (3H, m , H-5, and H-6), 5.60 (1H, dd , J = 8.1, 9.0 Hz, H-2), 5.63 (1H, t , J = 9.0 Hz, H-4), 6.00 (1H, t , J = 9.0 Hz, H-3), 6.27 (1H, d , J = 8.1 Hz, H-1), 6.91, 7.00, 7.01, 7.07, 7.13 ( 2H, s , galloyl H) each;

¹³ C-NMR (75 MHz, MeOD): 62.7 (C-6), 69.3 (C-4), 71.7 (C-2), 73.3 (C-3), 74.0 (C-5), 93.3 (C-1 ).

Experimental Example  1: Platelet aggregation inhibitory effect (In in vitro )

In order to determine the effect of inhibiting platelet aggregation of the lacquer bark extract of the present invention, fractions thereof and the effective single substance separated therefrom, the following experiments were performed.

1-1. Rat  Inhibitory effect on platelet aggregation in blood

Male Sprague-Dawley (SD) rats, 6-8 weeks of age, were purchased from Daehan Biolink and purified for 1 week with free water and feed. Blood was drawn from rats and immediately dispensed into a vaccutainer containing 3.2% sodium citrate anticoagulant to prevent coagulation. Platelet Rich Plasma (PRP) in the collected blood was centrifuged at 1100 rpm for 10 minutes and then the layers were recovered. The remaining layer was centrifuged at 3000 rpm for 20 minutes to further reduce platelet plasma (PPP). ). Platelet counts were counted using a hemocytometer (Coulter JT, USA), and platelet aggregation inhibitory activity was measured using a platelet aggregation analyzer (Chrono log, USA). After appropriate dilution with physiological saline to platelet count to 4 × 10 ⁸ cells / ㎖, and incubated for 10 minutes at 37 ℃, 50 μl of the fraction (sample suspension) of the lacquer extract prepared in the above Example was added for 5 minutes I was. The platelet aggregation inducer (collagen) was added in a certain amount and reacted at 37 ° C. with stirring at 1000 rpm to determine the degree of platelet aggregation as a change in impedance.

Samples were prepared by extracting the lacquer extract fraction prepared in the above example, suspended in 10% DMSO solution.

Saline was used as a negative control and aspirin was used as a positive control.

The results are shown in Tables 1-3.

Anti-platelet Aggregation Inhibitory Effect of the Extracts from the Bark Extracts of the Invention in Platelet-rich Plasma of Rats Induced by sample Concentration (µg / ml) Impedance % Inhibition Negative control group (physiological saline) - 15 ± 1.9 - Positive control group (Aspirin) 5 7.6 ± 2.3 46.5 ± 13.2 2.5 11 ± 2.6 22.6 ± 21.0 Methanol extract 250 4.5 ± 0.7 74.7 ± 5.9 200 5 73.6 150 8 57.8 100 17 10.5 50 23.5 ± 0.7 -31.1 ± 14.2 Ethyl acetate layer 250 1 ± 1 93.1 ± 7.8 200 1.5 ± 0.7 89.6 ± 7.1 150 2.5 ± 2.1 81.9 ± 18.0 100 5 ± 2.8 65.1 ± 26.9 50 15.3 ± 3.6 2.7 ± 43.4 Fraction 2 250 1.6 ± 0.5 88.1 ± 5.1 150 4.0 ± 2.8 69.7 ± 22.5 100 10.5 ± 0.7 21.9 ± 9.3 50 16.5 ± 2.1 -17.8 ± 15.1

As shown in Table 1, the platelet aggregation inhibition rate of the lacquer bark extract according to the present invention was 74.7 ± 5.9% at 250 μg / ml of methanol extract, and 93.1 ± 7.8% at 250 μg / ml of ethyl acetate layer. In the case of the positive control aspirin, the inhibition rate was 46.5 ± 13.2% at 5 μg / ml. Therefore, it can be seen that the platelet aggregation inhibitory activity is the best in the ethyl acetate layer.

In addition, among the fractions of the bark bark extract isolated from the ethyl acetate layer according to the present invention, the inhibition rate was high in fraction 2, and was proportional to the concentration. In case of fraction 1, it was impossible to measure due to the small amount of separation. In case of fractions 3, 4, and 5, insoluble solubility was not possible.

Therefore, it can be seen that the platelet aggregation inhibition rate is excellent in the ethyl acetate layer of the lacquer bark extract according to the present invention and fraction 2 separated therefrom.

1-2. Inhibitory Effects of Platelet Aggregation on Human Blood

In Experimental Example 1-1, the fraction 2 and the fraction thereof whose platelet aggregation inhibitory effect was confirmed in rat blood were measured as follows.

We randomly selected 11 adult females and 4 adult males between the ages of 20 and 50, and did not take aspirin or nonsteroidal anti-inflammatory drugs a week before blood collection.

At a given time, blood was drawn with a 10 ml disposable plastic syringe and then dispensed into a vactainer containing 3.2% sodium citrate anticoagulant to prevent coagulation. Platelet counts of whole blood were counted using a hemocytometer (Coulter JT, USA), and then diluted appropriately with physiological saline so that the platelet count was 4 × 10 ⁸ cells / ml. Since the method was the same as in Experimental Example 1-1. However, collagen, adenosine diphosphate (ADP), or arachidonic acid was used as a platelet aggregation inducer, and the experiment was repeated for each concentration using an effective single component separated from the lacquer extract as a sample. The platelet aggregation inhibitory ability is expressed as an IC ₅₀ value, which is the concentration of the effective single component corresponding to the inhibition rate according to the concentration or the platelet aggregation inhibition rate calculated by using the same.

The platelet aggregation inhibitory effect of Fraction 2 and Fractions 2-1 to 2-4 of the lacquer bark extract according to the present invention is shown in Tables 2 to 4.

Inhibition of Platelet Aggregation of Fraction 2 and Fractions 2-1 to 2-4 of the Bark Extract of the Present Invention in Human Whole Blood Induced by Collagen sample Concentration (µg / ml) Impedance % Inhibition Negative control group (physiological saline) - 14.1 ± 2.5 - Positive control group (aspirin) 500 6.5 ± 2.5 60.0 ± 13.9 Fraction 2 500 8.4 ± 4.5 43.3 ± 25.7 250 9.6 ± 2.3 34.3 ± 17.8 Fraction 2-1 500 10.0 ± 0.0 28.6 ± 0.0 Fraction 2-2 500 3.7 ± 0.6 77.8 ± 3.9 250 6.0 ± 2.8 60.0 ± 18.9 Fraction 2-3 500 4.5 ± 0.7 71.9 ± 4.4 250 14.0 ± 1.4 12.5 ± 8.8 Fractions 2-4 500 11.0 ± 00 21.4 ± 00 Isomalthol - - IC ₅₀ = 3.1 mM Pentagaloyl glucose - - IC ₅₀ = 1.6 × 10 ^-1 mM

Inhibitory Effects of Platelet Bark Extract Fraction 2 and Fractions 2-2 to 2-3 Fractions According to the Present Invention on Whole Blood of ADP-induced Human Platelets sample Concentration (µg / ml) Impedance % Inhibition Negative control group (physiological saline) - 7.8 ± 2.6 - Positive control group (aspirin) 500 4.7 ± 3.3 42.4 ± 31.8 Fraction 2 500 3.7 ± 3.3 66.4 ± 25.3 250 3.0 ± 2.0 48.5 ± 36.9 Fraction 2-2 500 3.8 ± 3.9 61.1 ± 37.6 250 3.7 ± 4.0 57.8 ± 36.7 Fraction 2-3 500 1.0 ± 0.0 87.1 ± 5.4 250 5.3 ± 5.9 62.2 ± 24.5 Isomalthol - - IC ₅₀ = 5.4 × 10 ^-1 mM Pentagaloyl glucose - - IC ₅₀ = 1.77 × 10 ^-1 mM

Inhibitory Effect of Aqueous Bark Extract Fraction 2 and Fractions 2-2 to 2-3 Induced Platelet Aggregation in Human Whole Blood Induced by Arachidonic Acid sample Concentration (µg / ml) Impedance % Inhibition Negative control group (physiological saline) - 9.9 ± 2.0 - Positive control group (aspirin) 500 0.25 ± 0.5 98.3 ± 3.5 Fraction 2 500 0.0 ± 0.0 100.0 ± 0.0 250 3.3 ± 3.5 62.5 ± 45.1 Fraction 2-2 500 0.25 ± 0.5 97.5 ± 5.0 250 3.7 ± 4.0 35.4 ± 41.1 Fraction 2-3 500 0.0 ± 0.0 100.0 ± 0.0 250 0.0 ± 0.0 100.0 ± 0.0 Isomalthol - - IC ₅₀ = 1.1 × 10 ^-1 mM Pentagaloyl glucose - - IC ₅₀ = 2.2 × 10 ^-1 mM

As shown in Tables 2 to 4, Fraction 2 of the bark extract according to the present invention showed a high platelet aggregation inhibition rate in whole blood of humans induced by agglutinants (collagen, ADP or arachidonic acid). In addition, when the collagen was used as a coagulant-inducing agent in the fractions separated therefrom, the platelet aggregation inhibition rate was high in fraction 2-2, and when the ADP or arachidonic acid was used as the coagulant-inducing agent in fraction 2-3, High. In addition, isomaltol and pentagalloyl glucose, which are effective single substances separated from the fractions, showed very low IC ₅₀ values, and thus the effect was very high.

Experimental Example  2 : Platelet receptor  Expression inhibitory effect (In in vitro )

Experimental Example 1 In order to determine the effect of inhibiting the platelet receptor expression of the effective single substance derived from the fraction of the lacquer bark extract of the present invention excellent in inhibiting platelet aggregation, the following experiment was performed.

Platelet receptor expression corresponds to CD41 (Anti-Human Platelet GPIIb / IIIa Complex, Becton Dickinson), PAC-1 (type 1 PACAP receptor, Becton Dickinson), P-selectin (P-selectin, Becton Dickinson) Antibodies were used and experimented using a flow cytometer (Becton Dickinson). 90 µl of human whole blood was incubated at room temperature for 5 minutes, 10 µl of sample suspension was added, and 10 µM of adenosine diphosphate (ADP) was added as an inducer of platelet activity. 20 μl of a monoclonal antibody corresponding to each of CD41, PAC-1, and P-selectin is added thereto, and incubated at room temperature in the dark for 20 minutes. 300 μl of 2% paraformaldehyde was added to stop the reaction, and then the degree of platelet receptor expression was measured by means of flow cytometry using MFI (Mean Fluorescence intensity) value, indicating that platelet receptor expression was inhibited by an effective single substance.

PBS buffer was used as a negative control and aspirin was used as a positive control.

The inhibitory effect of platelet receptor expression on the effective single substance isolated from the lacquer bark extract fraction according to the present invention is shown in Table 5.

Inhibitory Effect of Platelet Receptor Expression (MFI) on Effective Single Substances Isolated from the Bark Extract Fractions of the Present Invention in Human Whole Blood Induced by ADP Platelet receptor Negative Control (PBS) Positive control group (aspirin, 1000 μg / ml) Isomaltol (1000 μg / ml) Pentagaloyl glucose (500 μg / ml) CD41 1658.1 ± 348.52 1482.19 ± 144.59 1392.66 ± 328.53 1598.54 ± 269.33 PAC-1 67.37 ± 15.21 49.54 ± 0.99 * 40.83 ± 7.48 * 46.27 ± 0.51 * P-selectin 46.92 ± 6.92 37.3 ± 0.98 34.92 ± 1.94 33.21 ± 33.21 ± 3.01 *

* p <0.05

As shown in Table 5, inhibition of platelet receptor expression in whole blood of humans induced by ADP as a platelet activator was high in the effective single substance isolated from the fraction of the bark extract according to the present invention.

Experimental Example  3: Platelet Calcium Mobilization  Inhibitory Effect (In in vitro )

In order to examine the effects of inhibiting platelet aggregation and inhibiting platelet receptor expression in Experimental Example 1 and Experimental Example 2, an effective single substance derived from the fraction of the lacquer bark extract of the present invention, the inhibition of platelet calcium mobilization, The experiment was performed.

To test platelet calcium mobilization, fluorine-3AM (Fluo-3AM, Becton Dickinson) was used for calcium fluorescence staining, and a flow cytometer (Becton Dickinson) was used. Calcium fluorescence-stained fluoro-3AM was added to 1 ml of human platelet-rich plasma to a final concentration of 4 μM, and then incubated at 37 ° C. for 30 minutes. 20 µl of the effective single substance isolated from the fraction of the lacquer extract was added to 180 µl of the culture solution, followed by incubation for 5 minutes at 37 ° C, followed by addition of 300 µl of Tyros-Tyrode's buffer. 25 μl of ADP (final concentration 10 μM) was added thereto, and the degree of calcium mobilization was measured using a flow cytometer while gently shaking.

PBS buffer was used as a negative control and aspirin was used as a positive control.

The inhibitory effect of platelet calcium mobilization of the effective single substance isolated from the lacquer bark extract fraction according to the present invention is shown in Table 6.

Inhibitory Effect on Platelet Calcium Mobilization (MFI) of Effective Single Substances Isolated from the Bark Extract Fraction of the Present Invention Measurement period Negative Control (PBS) Positive control group (aspirin, 1000 μg / ml) Isomaltol (1000 μg / ml) Pentagaloyl glucose (500 μg / ml) Reaction electricity 4.58 ± 0.6 4.43 ± 0.38 4.43 ± 0.41 4.3 ± 0.35 Initial reaction 16.91 ± 1.76 13.25 ± 3.75 ** 13.16 ± 1.07 *** 13.82 ± 1.78 *** Middle reaction 12.01 ± 1.57 8.67 ± 2.46 ** 8.05 ± 0.61 *** 10.31 ± 1.71 ** End of reaction 10.6 ± 1.35 7.67 ± 1.9 *** 7.08 ± 0.35 *** 9.25 ± 1.58 *

*** p <0.001, ** p <0.01, * p <0.1

As shown in Table 6, the effective single substance isolated from the bark bark extract according to the present invention showed a lower MFI value than the negative control group, inhibiting platelet calcium mobilization in platelet-rich plasma of human induced by platelet activator ADP. The effect was confirmed to be high.

Experimental Example  4 : Antithrombotic  Effect (In vivo )

In order to examine the antithrombotic effect of the fraction of the lacquer bark extract of the present invention excellent in platelet aggregation inhibitory effect and platelet receptor receptor expression in Experimental Example 1 and Experimental Example 2, the following experiment was performed.

20-25 g of male ICR mice were orally administered fraction suspension or physiological saline of the present invention in proportion to body weight. A mixed solution containing collagen (1200 μg / kg) and epinephrine (120 μg / kg) in 100 μl of physiological saline was injected into the tail vein after about 1 hour and observed for 15 minutes of continued paralysis, death or recovery from paralysis. The antithrombotic effect of the sample was determined.

Saline was used as a negative control and aspirin was used as a positive control.

The results are shown in Table 7.

sample Dose (mg / kg) Number of deaths or paralysis / number of experiments Antithrombotic rate (%) Negative control group (physiological saline) - 8/10 20.0 Positive control group (aspirin) 10 2/9 66.7 Fraction 2 100 4/11 63.6

As shown in Table 7, when 100 mg / kg of fraction 2 of the extract of the bark bark extract according to the present invention exhibited an antithrombogenic rate of 63.6%, the antithrombotic rate was increased compared to the negative control.

Examples of preparations for the compositions of the present invention are illustrated below.

Formulation example  1: Preparation of Pharmaceutical Formulations

1-1: Powder  Produce

Lacquer bark extract according to the present invention, fractions thereof or effective single substance separated therefrom 2 g

1 g lactose

After mixing the above components, the airtight cloth was filled to prepare a powder.

1-2: Preparation of Tablets

Succulent bark extract, fractions thereof, or effective single substance isolated therefrom according to the present invention 100 mg

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

After mixing the above components, tablets were prepared by tableting according to a conventional method for producing tablets.

1-3: Preparation of Capsule

Succulent bark extract, fractions thereof, or effective single substance isolated therefrom according to the present invention 100 mg

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

After mixing the above components, the capsule was prepared by filling in gelatin capsules according to the conventional method for producing a capsule.

1-4: Preparation of Injection Solution

Sumac bark extract according to the present invention, fractions thereof or 10 ㎍ / ㎖ effective single substance separated therefrom

Dilute hydrochloric acid BP until pH 3.5

Injectable sodium chloride BP up to 1 ml

Dissolve the lacquer bark extract according to the present invention, fractions thereof or the effective single substance separated therefrom in a suitable volume of sodium chloride BP for injection, and adjust the pH of the resulting solution to pH 3.5 using dilute hydrochloric acid BP. The volume was adjusted using sodium chloride BP and mixed well. The solution was filled into a 5 ml Type I ampoule made of clear glass, encapsulated under an upper grid of air by dissolving the glass, and sterilized by autoclaving at 120 ° C. for at least 15 minutes to prepare an injection solution.

Formulation example  2: manufacture of food

Succulent bark extract according to the present invention, its fractions or foods containing an effective single material separated therefrom were prepared as follows.

2-1: Manufacturing Flour Food

0.1 to 10.0 parts by weight of lacquer bark extract according to the present invention, fractions thereof or an effective single substance separated therefrom is added to flour, and bread, cake, cookies, crackers and noodles are prepared by using the mixture in a conventional manner for health. Enhancement foods were prepared.

2-2: soup  And preparation of gravy

The lacquer bark extract according to the present invention, fractions thereof, or 0.1 to 1.0 parts by weight of an effective single substance separated therefrom was added to soups and broth to prepare meat products for health promotion, soups and broths in a conventional manner.

2-3: Ground Ground beef  Produce

10 parts by weight of the lacquer bark extract according to the present invention, fractions thereof or the effective single substance separated therefrom was added to the ground beef to prepare ground beef for health promotion in a conventional manner.

2-4: Manufacture of Dairy Products

The lacquer bark extract according to the present invention, fractions thereof, or 0.1 to 1.0 parts by weight of an effective single substance separated therefrom was added to milk, and various dairy products such as butter and ice cream were prepared in a conventional manner using the milk.

2-5: Linear  Produce

Brown rice, barley, glutinous rice, and yulmu were alphad by a known method, and then dried and roasted to prepare a powder having a particle size of 60 mesh.

Black beans, black sesame seeds, and perilla were also steamed and dried by a known method, and then ground to a powder having a particle size of 60 mesh.

The lacquer bark extract according to the present invention, fractions thereof or the effective single substance separated therefrom were reduced and concentrated in a vacuum concentrator, dried by spraying and drying with a hot air dryer, and then pulverized with a particle size of 60 mesh to obtain a dry powder.

Cereals, seeds and the extract of the lacquer bark according to the present invention, the fractions thereof or the dry powder of the effective single material separated therefrom were prepared in a conventional manner by combining in the following proportions.

Cereals (30 parts by weight brown rice, 15 parts by weight brittle, 20 parts by weight of barley),

Seeds (7 parts by weight perilla, 8 parts by weight black beans, 7 parts by weight black sesame seeds),

Succulent bark extract, fractions thereof, or dry powder of an effective single material separated therefrom according to the present invention (1 part by weight),

Ganoderma lucidum (0.5 parts by weight),

Foxglove (0.5 part by weight)

Formulation example  3: manufacture of beverage

Succulent bark extract according to the present invention, a fraction thereof or a beverage comprising an effective single substance separated therefrom was prepared as follows.

3-1: Health drink  Produce

Substances such as liquid fructose (0.5%), oligosaccharides (2%), sugars (2%), salts (0.5%), water (75%) and lacquer bark extracts according to the present invention, fractions thereof or effective separations therefrom After homogeneously blending a single substance and sterilizing it immediately, it was packaged in a small packaging container such as a glass bottle or a plastic bottle to prepare a health beverage.

3-2: Vegetable juice  Produce

0.5 g of the lacquer bark extract according to the present invention, fractions thereof or the effective single substance separated therefrom was added to 1,000 ml of a vegetable juice such as tomato or carrot to prepare a vegetable juice for health promotion in a conventional manner.

3-3: Fruit juice  Produce

0.1 g of the lacquer bark extract according to the present invention, fractions thereof or the effective single substance separated therefrom was added to 1,000 ml of fruit such as apples or grapes to prepare fruit juice for health promotion in a conventional manner.

The lacquer bark extract according to the present invention, its fractions and the effective single substance isolated therefrom are excellent in inhibiting platelet aggregation of rats induced by collagen as well as whole blood platelet aggregation in humans induced by various aggregation inducing agents in vitro. In addition, the anti-thrombotic effect of platelet receptor expression and platelet calcium mobilization as well as excellent anti-thrombotic effect in vivo, prevent the prevention of diseases involving thrombosis such as atherosclerosis, cerebral hemorrhage, stroke and cerebral infarction and It can be usefully used for treatment.

Claims (10)

An antithrombotic composition comprising a lacquer bark extract containing isomaltol or pentagaloyl glucose as an active ingredient. The anti-thrombotic composition according to claim 1, wherein the lacquer bark extract is extracted with water, C ₁ -C ₂ alcohol, ethyl acetate or a mixture thereof. The anti-thrombotic composition according to claim 2, wherein the C ₁ to C ₂ alcohol is methanol. The anti-thrombotic composition according to claim 2, wherein the lacquer bark extract is an ethyl acetate layer obtained by extracting with water, C ₁ -C ₂ alcohol or a mixture thereof and then extracting with ethyl acetate. The anti-blood composition according to claim 4, wherein the lacquer bark extract is a fraction obtained by separating the ethyl acetate layer extracted with water, C ₁ ~ C ₂ alcohol or a mixture thereof with ethyl acetate by column chromatography. . An anti-thrombotic composition comprising isomaltol or pentagalloyl glucose, which is an effective single substance, as an active ingredient. delete The anti-thrombotic composition according to any one of claims 1 to 6, wherein the anti-thrombotic composition is used for the prevention and treatment of diseases involving thrombosis. 9. The antithrombotic composition according to claim 8, wherein the disease involving thrombosis includes arteriosclerosis, cerebral hemorrhage, stroke and cerebral infarction. Platelet aggregation inhibitor containing isomaltol or pentagalloyl glucose as an active ingredient.

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