Antithrombotic composition containing <i>Polygonum cuspidatum sieb. et zucc. </i>and <i>Cinnamomum cassia blume</i>

Abstract

The present invention relates to an antithrombotic composition including Polygonum cuspidatum Sieb. et Zucc. and Cinnamomum cassia Blume as active ingredients, and preferably includes an extract obtained by mixing Polygonum cuspidatum Sieb. et Zucc. and Cinnamomum cassia Blume in a weight ratio of 1:2-2:1 and extracting the mixture. The composition of the present invention is highly effective in antithrombotic effects, identified as a platelet aggregation inhibitory effect, a thrombosis inhibitory effect, a thrombosis-delaying effect, and the like, and thus can be used as an effective herbal-medicine-based agent for preventing or treating thrombosis, an antithrombotic health food, and the like.

Claims

1. An antithrombotic composition comprising Polygonum cuspidatum Sieb. et Zucc. and Cinnamomum cassia Blume as active ingredients.

2. The antithrombotic composition according to claim 1, wherein the Polygonum cuspidatum Sieb. et Zucc. and the Cinnamomum cassia Blume are an extract obtained by mixing in a weight ratio of 1:2-2:1 and through extraction.

3. The antithrombotic composition according to claim 2, wherein the Polygonum cuspidatum Sieb. et Zucc. and the Cinnamomum cassia Blume are an extract obtained by mixing in a weight ratio of 1:1 and through extraction.

4. The antithrombotic composition according to claim 2, wherein the extract is obtained by adding an extraction solvent at a volume that is 150 times to 250 times that of the mixture of the Polygonum cuspidatum Sieb. et Zucc. and the Cinnamomum cassia Blume, followed by immersion therein at 50° C. to 70° C. for 22 hours to 26 hours, thereby obtaining an extract, filtering the extract, concentrating the filtrate under reduced pressure, and drying the concentrate.

5. The antithrombotic composition according to claim 4, wherein the solvent is 70% ethanol.

6. An agent for preventing or treating thrombosis, the agent comprising the composition of claim 1 as an active ingredient.

7. An antithrombotic health food comprising the composition of claim 1.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 illustrates GC-MS analysis results of confirming the extracted components of a mixed extract of Polygonum cuspidatum Sieb. et Zucc. and Cinnamomum cassia Blume.

(2) FIG. 2 illustrates the results of confirming the inhibitory effect of a composition of the present invention on a platelet aggregation phase induced by collagen.

(3) FIGS. 3 and 4 illustrate the results of confirming the effect of a composition of the present invention on delaying thrombosis in a thrombogenic animal model.

(4) FIG. 5 illustrates the results of confirming histological changes in blood vessels in a thrombogenic animal model.

DETAILED DESCRIPTION OF THE INVENTION

(5) An antithrombotic composition of the present invention includes, as active ingredients, Polygonum cuspidatum Sieb. et Zucc. and Cinnamomum cassia Blume, and preferably includes an extract obtained by mixing Polygonum cuspidatum Sieb. et Zucc. and Cinnamomum cassia Blume in a weight ratio of 1:2-2:1 and extracting the mixture. Particularly preferably, the composition includes an extract obtained by mixing Polygonum cuspidatum Sieb. et Zucc. and Cinnamomum cassia Blume in a weight ratio of 1:1 and extracting the mixture.

(6) It is preferable to use water or ethanol as a solvent for extraction. It is particularly preferable to use 70% ethanol as an extraction solvent. When 70% ethanol is used as the extraction solvent, more components are extracted than when water is used as the extraction solvent (see FIG. 1).

(7) In addition, the extract obtained by mixing Polygonum cuspidatum Sieb. et Zucc. and Cinnamomum cassia Blume and extracting the mixture includes all components of an extract obtained by extracting each of Polygonum cuspidatum Sieb. et Zucc. and Cinnamomum cassia Blume, and also additionally includes other components (see FIG. 1).

(8) The extraction of Polygonum cuspidatum Sieb. et Zucc. and Cinnamomum cassia Blume may be performed using a general extraction method, and examples of a preferable extraction method are as follows.

(9) An extraction solvent is added to the mixture of Polygonum cuspidatum Sieb. et Zucc. and Cinnamomum cassia Blume at a volume that is 150 times to 250 times that of the mixture, the mixture is immersed therein at 50° C. to 70° C. for 22 hours to 26 hours, and then an extract is obtained at room temperature. This process may be repeated two or three times. After filtering the extract, the filtrate is concentrated under reduced pressure and dried to obtain a mixed extract of Polygonum cuspidatum Sieb. et Zucc. and Cinnamomum cassia Blume.

(10) The antithrombotic composition of the present invention is highly effective in antithrombotic effects, identified as a platelet aggregation inhibitory effect, a thrombosis inhibitory effect, a thrombosis-delaying effect, and the like. In particular, compared to the case of using either Polygonum cuspidatum Sieb. et Zucc. or Cinnamomum cassia Blume alone, the effect is much stronger, and the thrombosis-delaying effect is about twice as strong as that of cardiotonic pills, which are currently commercially available as an antithrombotic medicinal herb composition. Therefore, the antithrombotic composition of the present invention may be used as an effective herbal-medicine-based agent for preventing or treating thrombosis, an antithrombotic health food, and the like.

(11) A dried extract of Polygonum cuspidatum Sieb. et Zucc. and Cinnamomum cassia Blume of the antithrombotic composition of the preset invention may be used in an amount of 100 mg to 1,000 mg daily based on an adult body weight of 60 kg when used for the prevention of thrombosis, and in an amount of 500 mg to 5,000 mg daily when used for the treatment of thrombosis.

(12) Hereinafter, the present invention will be described in further detail with reference to the following examples. These examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention.

EXAMPLES

Example 1

(13) Preparation of Mixed Extract of Polygonum cuspidatum Sieb. et Zucc. and Cinnamomum cassia Blume

(14) 20 l of 70% ethanol was added to 100 g of Polygonum cuspidatum Sieb. et Zucc. and 100 g of Cinnamomum cassia Blume, which were purchased from Human Herb (http://www.humanherb.co.kr/), followed by immersion therein at 60° C. for 24 hours, and then an extract was obtained at room temperature. Again, 20 l of 70% ethanol was added, and the extraction process was repeated twice more to collect the extract.

(15) The filtrate obtained by filtering each extract was concentrated under reduced pressure by evaporating the solvent in a vacuum rotary evaporator (Nihon Seiko, Japan, VR-205c) and dried to obtain 41.8 g of a mixed extract of Polygonum cuspidatum Sieb. et Zucc. and Cinnamomum cassia Blume (yield: 20.9%).

Example 2

(16) Preparation of Mixed Extract of Polygonum cuspidatum Sieb. et Zucc. and Cinnamomum cassia Blume

(17) A 1:2 mixed extract of Polygonum cuspidatum Sieb. et Zucc. and Cinnamomum cassia Blume was obtained in the same manner as in Example 1, except that 66.7 g of Polygonum cuspidatum Sieb. et Zucc. and 133.3 g of Cinnamomum cassia Blume were used.

Example 3

(18) Preparation of Mixed Extract of Polygonum cuspidatum Sieb. et Zucc. and Cinnamomum cassia Blume

(19) A 2:1 mixed extract of Polygonum cuspidatum Sieb. et Zucc. and Cinnamomum cassia Blume was obtained in the same manner as in Example 1, except that 133.3 g of Polygonum cuspidatum Sieb. et Zucc. and 66.7 g of Cinnamomum cassia Blume were used.

Experimental Example 1

(20) Experiment for Platelet Aggregation Inhibitory Activity

(21) The aggregation of rat platelets was induced using collagen, and the ability of the composition of the present invention to inhibit platelet aggregation depending on the mixing ratio of Polygunum cuspidatum Sieb. et Zucc. and Cinnamomum cassia Blume was compared as follows.

(22) In the experiment, SD rats (Sprague-Dawley rats, male, 220 g) supplied from KOATECH were used.

(23) The SD rats had an acclimation period of 1 week, and then blood was collected from the abdominal aorta. 2 ml of thyroid buffer (137 mM NaCl, 12 mM NaHCO.sub.3, 5.5 mM glucose, 1 M MgCl.sub.2, 1 M KCl, and 1 M Na.sub.2HPO.sub.4, pH 7.4) was added to 5 ml of the collected blood, followed by centrifugation at 1,000 rpm for 10 minutes.

(24) The supernatant (platelet rich plasma; PRP) from which blood cells had been removed was centrifuged at 800×g for 15 minutes, and the precipitated platelets were washed twice with washing buffer (137 mM NaCl, 2.9 mM KCl, 1 mM MgCl.sub.2, 5 mM glucose, 12 mM NaHCO.sub.3, 0.34 mM Na.sub.2HPO.sub.4, 1 mM EDTA, 20 mM HEPES, and 0.25% BSA, pH 7.4) and then suspended with a suspension buffer (137 mM NaCl, 2.9 mM KCl, 1 mM MgCl.sub.2, 5 mM glucose, 12 mM NaHCO.sub.3, 0.34 mM Na.sub.2HPO.sub.4, 20 mM HEPES, and 0.25% BSA, pH 7.4) to thereby obtain final washed platelets. The obtained washed platelets were diluted to 3×10.sup.8 platelets/ml and used for analysis.

(25) The platelets were incubated at 37° C. for 3 minutes, 1 mM CaCl.sub.2 was then added thereto, and then 100 μg/ml of each of the sample of Example 1 (1:1 mixed extract of Polygonum cuspidatum Sieb. et Zucc. and Cinnamomum cassia Blume), the sample of Example 2 (1:2 mixed extract of Polygonum cuspidatum Sieb. et Zucc. and Cinnamomum cassia Blume), and the sample of Example 3 (2:1 mixed extract of Polygonum cuspidatum Sieb. et Zucc. and Cinnamomum cassia Blume) was added thereto, followed by incubation for 2 minutes.

(26) After incubation, 2.5 μg/ml of collagen, which is an inducer for platelet aggregation, was added to measure the degree of platelet aggregation for 5 minutes. The rat platelet aggregation inhibitory capacity was confirmed by a turbidity measurement method using an aggregometer (Chrono-Log Co., Ltd., Havertown, Pa. USA). The results thereof are illustrated in FIG. 2.

(27) As can be seen from the results of FIG. 2, all of the samples of Examples 1 to 3 exhibited excellent platelet aggregation inhibitory capacity, i.e., 63% or more. In particular, the sample of Example 1 (92.8±5.9%), which is a 1:1 mixed extract of Polygunum cuspidatum Sieb. et Zucc. and Cinnamomum cassia Blume, exhibited a very potent platelet aggregation inhibitory capacity compared to the sample of Example 2 (63.3±3.1%), which is a 1:2 mixed extract of Polygunum cuspidatum Sieb. et Zucc. and Cinnamomum cassia Blume, and the sample of Example 3 (72.0±4.4%), which is a 2:1 mixed extract of Polygunum cuspidatum Sieb. et Zucc. and Cinnamomum cassia Blume.

Experimental Example 2

(28) Confirmation of Effect on Blood Flow Changes in Carotid Artery Thrombus-Induced Animal Model

(29) The effects of a Polygunum cuspidatum Sieb. et Zucc. extract, a Cinnamomum cassia Blume extract, and a mixed extract of Polygunum cuspidatum Sieb. et Zucc. and Cinnamomum cassia Blume on blood flow changes in an animal model with carotid artery thrombi induced using ferric chloride (FeCl.sub.3) were measured as follows.

(30) As an experimental animal, male SD rats (Sprague-Dawley rats, 8 weeks old, 230-250 g, KOATECH, Korea) were purchased, had an acclimation period of 1 week, and were then used for the experiment.

(31) 1. Comparison between Single Extract and Mixed Extract

(32) As a sample, the mixed extract of Polygonum cuspidatum Sieb. et Zucc. and Cinnamomum cassia Blume of Example 1 was used. As samples for comparison, a Polygonum cuspidatum Sieb. et Zucc. extract and a Cinnamomum cassia Blume extract were used. The Polygunum cuspidatum Sieb. et Zucc. extract and the Cinnamomum cassia Blume extract were obtained in the same manner as in Example 1, except that 200 g of Polygunum cuspidatum Sieb. et Zucc. and 200 g of Cinnamomum cassia Blume were respectively used.

(33) Each sample was orally administered to experimental animals at doses of 50 mg, 100 mg, and 200 mg per 1 kg of body weight for 3 days, and as a control, the same amount of saline was administered. During the test period, experimental animals were allowed to freely eat solid feed and drink water, and the breeding environment was automatically maintained at a temperature of 23±0.5° C. and a relative humidity of 50±50% under 12 hour light/dark cycles.

(34) To anesthetize the experimental animals, an anesthetic in which rompun and Zoletil were mixed at a ratio of 2:3 was used.

(35) To induce carotid artery thrombi in the experimental animals, filter paper (2×2 mm) soaked with a 30% iron chloride solution was brought into contact with the carotid artery for 3 minutes, followed by removal of the filter paper and wiping with physiological saline, and then blood flow was measured using a blood flow measurer (Laser Doppler Flowmetry (LDF); BFL21, Transonic Instrument, USA) equipped with a probe (Powerlab/8sp, ADInstruments Pty Ltd, Castle Hill, NSW, Australia).

(36) After treatment with iron chloride, thrombosis was based on the time at which a blood measurement value dropped near zero, and measured up to 40 minutes in the case of a normal control. The results thereof are illustrated in FIG. 3.

(37) In the normal group (C Basal), no thrombus was produced within a total observation time of 40 minutes, and the group treated with iron chloride was found to have a blood flow value of 0, 17 minutes after treatment with iron chloride.

(38) The group (PC) administered with the Polygonum cuspidatum Sieb. et Zucc. extract and the group (CC) administered with the Cinnamomum cassia Blume extract exhibited a blood flow value corresponding to about 40% of that of the normal group 40 minutes after treatment with iron chloride, whereas the group (Mix) administered with the mixed extract of Polygunum cuspidatum Sieb. et Zucc. and Cinnamomum cassia Blume maintained a blood flow value corresponding to about 70% of that of the normal group.

(39) As such, it was confirmed that the extract obtained by mixing Polygunum cuspidatum Sieb. et Zucc. and Cinnamomum cassia Blume and extracting the mixture exhibited a significantly strong antithrombotic effect compared to a Polygunum cuspidatum Sieb. et Zucc. extract or Cinnamomum cassia Blume extract alone.

(40) 2. Comparison between Mixed Extract and Commercially Available Drug

(41) To compare the effect of the mixed extract of Polygunum cuspidatum Sieb. et Zucc. and Cinnamomum cassia Blume on a blood flow change, with that of a commercially available drug, an experiment was conducted as follows.

(42) As a commercially available drug, cardiotonic pills (17.5 mg of Salvia miltiorrhiza, 3.4 mg of Panax notoginseng, and 0.2 mg of borneol) were used.

(43) An experiment was conducted in the same manner as described above, except that the mixed extract of Polygonum cuspidatum Sieb. et Zucc. and Cinnamomum cassia Blume and cardiotonic pills were orally administered to experimental animals at a dose of 200 mg/kg (body weight). The results thereof are illustrated in FIG. 4.

(44) As can be confirmed in FIG. 4, both the group (Mix) administered with the mixed extract of Polygonum cuspidatum Sieb. et Zucc. and Cinnamomum cassia Blume and the group administered with cardiotonic pills exhibited a remarkable thrombosis-delaying effect, and particularly, the group (Mix) administered with the mixed extract of Polygonum cuspidatum Sieb. et Zucc. and Cinnamomum cassia Blume exhibited a thrombosis-delaying effect that was approximately twice that of the group administered with cardiotonic pills. Such a thrombosis inhibitory effect of the mixed extract of Polygunum cuspidatum Sieb. et Zucc. and Cinnamomum cassia Blume was found to be similar to that of the normal group.

Experimental Example 3

(45) Confirmation of Histological Changes in Blood Vessels in Thrombogenic Animal Model

(46) Histological changes in blood vessels of a thrombogenic animal model were confirmed as follows.

(47) After measuring thrombosis in Experimental Example 2, the carotid artery site where the thrombus was generated was extracted to a size of 3 mm to 4 mm.

(48) The extracted carotid artery site was fixed in 10% neutral paraformaldehyde for 24 hours, embedded in paraffin through a normal tissue treatment process, and then sectioned (4 μm thick). The produced sections were stained with hematoxylin & eosin to prepare a tissue specimen for an optical microscope.

(49) The specimen was observed using an optical microscope, and the results thereof are shown in FIG. 5.

(50) As can be confirmed in FIG. 5, the blood vessel of the group treated with iron chloride (FeCl.sub.3) was completely filled with a blood clot. In contrast, the group (FeCl.sub.3+Mix) administered with the mixed extract of Polygonum cuspidatum Sieb. et Zucc. and Cinnamomum cassia Blume exhibited reduced thrombosis compared to the group ((FeCl.sub.3+PC) administered with a Polygunum cuspidatum Sieb. et Zucc. extract or the group (FeCl.sub.3+CC) administered with a Cinnamomum cassia Blume extract.