JP2016044134A - Thrombus preventive therapeutic agent and method for producing the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for producing a thrombus preventive therapeutic agent that is used for the treatment of thrombotic diseases from an extract from Pterocarpus marsupium.SOLUTION: The present invention provides a method for producing a thrombus preventive therapeutic agent comprising an extract obtained by the steps of (1) powdering Pterocarpus marsupium plants, (2) extracting an extract from powder obtained in step (1) with an alcohol solution, and (3) extracting an extract that can be dissolved in ethyl acetate from extraction powder obtained in step (2).SELECTED DRAWING: None

Description

  The present invention relates to a thrombus preventive / therapeutic agent and a method for producing the same.

  Thrombus occurs when blood in a blood vessel forms a clot for some reason and damages the blood vessel wall. Thrombophlebitis, venous thromboembolism (economy class syndrome), arterial thromboembolism Diseases, disseminated intravascular coagulation syndrome (DIC) and the like are known. So far, drugs for treating thrombi have been developed, and stilbene derivatives are known as one of the active ingredients used in thrombosis therapeutic agents.

  In Patent Document 1, resveratrol, which is a stilbene derivative and has an antithrombotic effect, is extracted from a material consisting of one or a plurality of grape fruits, pericarps, seeds, shoots or stems using a mixed solvent of water and ethanol. How to do is described.

  However, resveratrol has a low absorption rate and retention rate in the body and a short residence time in blood. Therefore, in recent years, there has been a demand for a therapeutic agent that has a higher absorption rate / retention rate in the body than resveratrol and makes blood clot prevention more effective by increasing the residence time in blood. Therefore, the present inventors have discovered that a component extracted from plant Indian mushrooms can be used as an agent for preventing or treating thrombosis, leading to the present invention.

JP 2012-41296 A

  The present invention provides a thrombus preventive / therapeutic agent having a higher therapeutic effect than before and a method for producing the same by using an extracted component obtained by alcohol extraction from the whole tree of Indian mushroom (Pterocarpus marsupium).

  The invention according to claim 1 is obtained by pulverizing plant Indian mushroom (Pterocarpus marsupium), performing heat extraction from the powder with an alcohol solution, and pulverizing the extract to extract an extract that is soluble in ethyl acetate. The present invention relates to a thrombus preventive / therapeutic agent comprising the extract obtained.

  The invention according to claim 2 relates to the thrombus preventive and therapeutic agent according to claim 1, wherein the main component of the extract soluble in ethyl acetate is pterostilbene represented by the following formula (1).

The invention according to claim 3 includes (1) a step of pulverizing plant Indian mushroom (Pterocarpus marsupium), (2) a step of extracting an extract from an alcohol solution from the powder obtained in (1), and (3) The present invention relates to a method for producing a thrombus preventive / therapeutic agent comprising an extract obtained by extracting an extract soluble in ethyl acetate from the extracted powder obtained in (2).

  The invention according to claim 4 is characterized in that the main component of the extract soluble in ethyl acetate is pterostilbene represented by the following formula (1): About.

  The invention according to claim 5 relates to the method for producing a thrombus prevention and treatment agent according to claim 3, wherein the alcohol contained in the alcohol solution is selected from the group consisting of methanol, ethanol and propanol.

  According to the invention which concerns on Claim 1 and 3, the effective extract as a thrombus prevention therapeutic agent can be selectively obtained from a plant Indian mushroom through processes (1)-(3). Furthermore, since this extract can reduce the viscosity in blood and suppress the generation of thrombus, it can provide a thrombus preventive / therapeutic agent effective for the treatment of thrombotic diseases.

  According to the inventions according to claims 2 and 4, since the main component of the extract soluble in ethyl acetate is pterostilbene, it is possible to provide a thrombus prevention and treatment agent that is more effective in treating thrombotic diseases.

  According to the invention of claim 5, by using methanol, ethanol, or propanol as the alcohol solution, it is possible to extract a component effective for a thrombus therapeutic agent from Indian mushrooms.

It is a figure which shows the component of the extract (powder) extracted using the alcohol solution by the high performance liquid chromatograph (HPLC) in a present Example. It is a figure which shows the component of the extract soluble in ethyl acetate by the high performance liquid chromatograph (HPLC) in a present Example.

The agent for preventing or treating a thrombus according to the present invention is obtained through the following steps.
(1) Step of pulverizing plant Indian mushroom (Pterocarpus marsupium) (2) Step of extracting an extract from the powder obtained by (1) with an alcohol solution (3) Ethyl acetate from the extracted powder obtained by (2) Of extracting extract soluble in water

The method for producing a thrombus prevention / treatment agent according to the present invention will be described in detail.
First, step (1) will be described.
Plant Indian mushrooms are pulverized with a crusher or the like.
Indian mushroom is Pterocarpus marsupium belonging to the leguminous genus Citan, and is an evergreen tree distributed in southern Sri Lanka from India. At this time, the whole tree including Indian mushroom trunk (heartwood, sapwood, bark), branches, leaves, flowers, roots may be used, or Indian mushroom trunk, branches, leaves, flowers, roots. Any of or a combination thereof may be used. Moreover, it does not specifically limit about the particle size of the powder obtained by pulverization.

Step (2) will be described.
A component soluble in the alcohol solution is obtained from the powdered Indian mushroom powder using a mixed solvent (alcohol solution) composed of water and alcohol. As the alcohol used at this time, methanol, ethanol, propanol or the like is preferably used. Furthermore, the content of alcohol contained in the alcohol solution is preferably 30 to 100%. Also, if the alcohol content is less than 30%, it is difficult to extract pterostilbene, which is not preferable. By using an alcohol solution for this operation, it is possible to extract a component effective for the treatment of thrombotic diseases contained in Indian mushrooms. Furthermore, the mass ratio of the powdered Indian mushroom and the alcohol solution used for this extraction operation is preferably 1: 5-20.

  The solution containing the component soluble in the alcohol solution is concentrated to obtain a concentrated solution. By this operation, the concentration of the component of Indian mushroom dissolved in alcohol which is a component having a lower boiling point than water can be increased. The concentration temperature for this operation is preferably 40-60 ° C. The reason for this is to prevent decomposition of the components. Moreover, since there exists a possibility that solids, such as a powder, may be contained in the extract after concentration, it is preferable to remove by filtration.

Step (3) will be described.
Extracted powder (solid extract) is obtained by drying the concentrated solution obtained in the step (2) under reduced pressure using a vacuum dryer or the like. At this time, the apparatus used for vacuum drying is not particularly limited.

  Water and ethyl acetate are added to the obtained extracted powder, and components soluble in ethyl acetate are extracted from this extracted powder. Thereafter, the target extract is obtained by column chromatography using silica gel. This extract contains pterostilbene represented by the following formula (1) as a main component (see FIGS. 1 and 2 and described in detail in [0040] below).

  In the step (3), ethyl acetate may be added in a plurality of times to extract a component (target extract) that is soluble in ethyl acetate contained in water.

  The extract obtained by the above-described method can reduce the viscosity in blood and suppress the generation of thrombus, and thus can be used as a thrombus prevention / treatment agent effective in the treatment of thrombotic diseases.

  The effects of the present invention will be made clearer by showing examples relating to the thrombus preventing / treating agent and the method for producing the same according to the present invention. However, the present invention is not limited to the examples.

<Preparation of antithrombotic agent>
50 g of whole Indian mushroom tree (including trunk, branches, leaves, flowers, roots) was pulverized using a grinder. 50 g of the obtained powder and 500 ml of 50% ethanol solution were added to the distillation flask of the distillation apparatus (heat source (gas burner), distillation flask, T-tube, thermometer, cooler, cooling water, flask for storing the distillate). In addition, the temperature was set to 90 ° C., a component soluble in a 50% ethanol solution was obtained from Indian mushroom, and the liquid was concentrated by an evaporator. This is an ethanol extract. The extract concentrated by distillation under reduced pressure was dried under reduced pressure (40 ° C.) to obtain an extract powder.
To the separatory funnel, 500 mg of the extracted powder, 50 ml of water, and 40 ml of ethyl acetate were added to extract components soluble in ethyl acetate. At this time, ethyl acetate was added in three portions of 40 ml (total amount of 120 ml). Thereafter, an ethyl acetate extract (thrombus prevention / treatment agent) was obtained by concentration under reduced pressure. At this time, water added to the separatory funnel was used as a water extract.

<Component analysis>
The extracted powder and the components of the ethyl acetate extract in this example were analyzed by liquid chromatography.
The liquid chromatograph measuring apparatus and conditions used in this example are shown below.
Measuring instrument: HPLC system High-performance liquid chromatograph manufactured by Shimadzu Corporation
Column: COSMOSIL 5C18-MS-II (4.6 × 250 mm)
Column temperature: 40 ° C
Mobile phase: A phase was 30% methanol solution containing 0.1% phosphoric acid, and B phase was methanol.
The solution was fed for 30 minutes so that A / B = (100/0) → A / B = (0/100).
Liquid feeding speed: 0.8 ml / min
Detection: UV (290 nm)
The results are shown in FIGS.

<Blood rheology improvement test using endotoxin (LPS) -induced DIC pathological rats>
For each sample, male rats (8 weeks old, 7 animals per group) preliminarily raised for 1 week were used.
(Control)
The control in this study is healthy male rats. The rats were orally administered with 0.2% sodium carboxymethylcellulose (CMC • Na) once a day for 7 consecutive days. After administration on day 7, blood was collected from the abdominal vena cava under pentobarbital anesthesia.
(Carrier control)
The carrier control in this study is brought to LPS-induced DIC pathology and does not administer ingredients that are effective in preventing thrombus. Male rats were orally administered with 0.2% sodium carboxymethylcellulose (CMC · Na) once a day for 7 days. One hour after subject administration on the seventh day, LPS was administered under mild anesthesia with pentobarbital (a hypnotic). Four hours after the administration of LPS, blood was collected from the abdominal vena cava under pentobarbital anesthesia.
(Positive control)
As a positive control in this test, a drug known to be effective in preventing thrombus is administered. Male rats received heparin as a positive control 1 hour before LPS administration on day 7. Four hours after the administration of LPS, blood was collected from the abdominal vena cava under pentobarbital anesthesia.

Example 1
A subject prepared with an ethyl acetate extract of 50 mg / kg was orally administered once a day for 7 days. One hour after subject administration on the seventh day, LPS was administered under mild anesthesia with pentobarbital (a hypnotic). Four hours after the administration of LPS, blood was collected from the abdominal vena cava under pentobarbital anesthesia.
(Example 2)
Example 1 is the same as Example 1 except that a test sample prepared with an ethyl acetate extract of 200 mg / kg was used.
(Example 3)
Example 1 is the same as Example 1 except that a sample prepared with an ethyl acetate extract of 500 mg / kg was used.

The collected blood was subjected to biochemical tests for the number of platelets, white blood cells, red blood cells, blood fibrinogen, and fibrin degradation product (FDP) in the blood.
The whole blood passage time was obtained by using blood collected from rats of each sample of Examples, Controls, Carrier Controls, and Positive Controls. The measurement was performed using a blood fluidity measuring apparatus (MC-FAN: manufactured by Hitachi, Ltd.) loaded with 5 .mu.m, 8736 parallel).
50 μL of a mixture of 1 volume of 3.8% sodium citrate solution was applied to MC-FAN for each 9 volumes of blood collected from rats of Examples, Controls, Carrier Controls, and Positive Controls. Whole blood passage time of 50 μL was measured under 20 cm water column pressure. The measured value of the whole blood passage time was converted into the case where the passage time of the physiological saline was 12.0 seconds by the following formula using the passage time of 100 μL of the physiological saline measured immediately before.
Whole blood passage time = blood passage time (s) × 12.0 (s) / saline passage time (s)
The results are shown in Tables 1 and 2.

<Platelet aggregation inhibition test>
(Preparation of PRP suspension)
Blood was collected from the heart of a male rabbit (14 weeks old) at a ratio of 1 volume of 3.8% sodium citrate solution to 9 volumes of blood, then placed in a silicon tube and centrifuged (170 g, 10 minutes, 25 ° C.). Upper platelet rich plasma (PRP) was obtained. The obtained PRP was put in another silicon tube, capped and stored at room temperature. Further, the lower layer was centrifuged (1520 × g, 15 minutes, 25 ° C.) to obtain an upper layer platelet poor plasma (PPP). PRP was diluted with PPP so that the platelet count was 2.5-3.5 × 10 ⁵ / μL to obtain a PRP suspension. The PPP was set to the standard value of 100% permeability, and the PRP suspension was set to the standard value of 0% permeability.

(Preparation of test solution)
Positive control In this test, a drug having an inhibitory effect on aggregation was used as a positive control. Indomethacin was used as a positive control drug. After indomethacin was dissolved in dimethyl sulfoxide (DMSO), DMSO was added with 50 mM Tris-HCl buffer (Tris-HCl buffer, buffer adjusted to pH 7.4 with 1 M HCl solution). What was dissolved so that the density | concentration of 0.5% might be used.

Negative control The negative control in this test was prepared by dissolving 50 mM Tris-HCl buffer so that the concentration of DMSO was 0.5% without administering an effective component for aggregation suppression.

Example : An extract composed of components dissolved in an ethanol solution from an Indian mushroom powder (ethanol extract), an extract composed of components soluble in ethyl acetate (ethyl acetate extract), and a component soluble in water Each extract (water extract) was dissolved in dimethyl sulfoxide (DMSO), and then DMSO with 50 mM Tris-HCl buffer (buffer adjusted to pH 7.4 with Tris-HCl buffer and 1 M HCl solution). What was dissolved so that the density | concentration of 0.5% might be used.

Comparative Example A similar test was also conducted on pterostilbene and resveratrol, which are conventionally known to have an aggregation-inhibiting effect. Pterostilbene and resveratrol were dissolved in dimethyl sulfoxide (DMSO), and then dissolved in 50 mM Tris-HCl buffer so that the concentration of DMSO was 0.5%.

(Aggregation inhibition test)
200 μL of the prepared PRP was dispensed into a cuvette dedicated to an aggregometer (PRP313M, MC Medical Inc.) and stirred at 37 ° C. and 1000 rpm for 3 minutes, and then 11 μL of the test solution was added using a microsyringe. The mixture was further stirred at 37 ° C. and 1000 rpm for 3 minutes, and then 11 μL of collagen (100 μg / mL, dissolved in a diluent) was added as a flocculant using a microsyringe. The change in permeability of the PRP suspension caused by platelet aggregation was recorded using an aggregometer for 10 minutes after addition of the flocculant. Platelet aggregation rate (%) was evaluated as an increase in maximum permeability in 10 minutes after addition of the aggregating agent. The permeability was set to 0% for PRP suspension and 100% for PPP. The platelet aggregation rate of the subject was defined as the reduction rate of the maximum permeability of the subject with respect to the maximum permeability of the control group.
The results are shown in Tables 3 and 4.

<Results of preparation of thrombus preventive and therapeutic agent>
1 and 2, the vertical axis represents peak intensity, and the horizontal axis represents retention time. At this time, the peak indicated by the retention time 28.14 [min] is a peak of pterostilbene.
As shown in FIG. 1, an extract containing pterostilbene as a main component could be extracted from Indian mushroom powder by using an ethanol solution as an extract. Further, as shown in FIG. 2, since the peak indicating pterostilbene is higher than that in FIG. 1, by further fractionating the extract obtained by ethanol distillation from the extracted powder with ethyl acetate, An extract could be obtained selectively.

<Blood rheology improvement test results>
As shown in the Examples of Table 1, it can be seen that the whole blood passage time is shortened as the dose of the target extract increases. From this result, it was found that an ethyl acetate extract (target extract) extracted from Indian mushrooms using an alcohol solution has a function of reducing blood viscosity.

<Platelet aggregation inhibition test results>
As shown in Table 3, an extract extracted from an ethanol solution (ethanol extract) and an extract obtained from a component soluble in ethyl acetate (ethyl acetate extract) have a low rate of platelet aggregation. I understand.
Resveratrol and pterostilbene are known to have an effect of lowering the platelet aggregation rate. As shown in Table 4, although pterostilbene alone is effective, the ethyl acetate extract (target extract) extracted from Indian mushroom is more effective. This is presumably because the extract extracted from Indian mushroom contains components other than pterostilbene that have the effect of reducing the aggregation rate.

  The extract obtained by extracting Indian mushrooms by the thrombus preventing / treating agent and the method for producing the same according to the present invention is suitably used for the thrombus preventing / treating agent in thrombotic diseases.

Claims (5)

  Thrombus prevention treatment comprising an extract obtained by pulverizing plant Indian mushroom (Pterocarpus marsupium), performing heat extraction from the powder with an alcohol solution, and extracting the extract soluble in ethyl acetate by pulverizing the extract Agent. The thrombus preventive and therapeutic agent according to claim 1, wherein the main component of the extract soluble in ethyl acetate is pterostilbene represented by the following formula (1).
(1) a step of pulverizing plant Indian mushroom (Pterocarpus marsupium), (2) a step of extracting an extract with an alcohol solution from the powder obtained by (1), and (3) extraction obtained by (2) A method for producing a thrombus preventive / therapeutic agent comprising an extract obtained by extracting an extract soluble in ethyl acetate from a powder. The method for producing a thrombus-preventing / treating agent according to claim 3, wherein the main component of the extract soluble in ethyl acetate is pterostilbene represented by the following formula (1).
  The method for producing a thrombus prevention and treatment agent according to claim 3, wherein the alcohol contained in the alcohol solution is selected from the group consisting of methanol, ethanol and propanol.