JP6523634B2 - Thrombus preventive and therapeutic agent and method for producing the same - Google Patents

Description

  The present invention relates to an antithrombotic therapeutic agent and a method for producing the same.

  Thrombosis is caused by blood clots in blood vessels forming a lump and injury to the blood vessel wall mainly, and thrombophlebitis, venous thromboembolism (economy class syndrome), arterial thromboembolism as diseases caused by thrombi. And disseminated intravascular coagulation syndrome (DIC) are known. Until now, drugs for treating thrombus have been developed, and stilbene derivatives are known as one of the active ingredients used for thrombus treating agents.

  In Patent Document 1, resveratrol, which is a stilbene derivative and has antithrombotic activity, is extracted from a material comprising one or more of grape fruits, peels, seeds, shoots or stems using a mixed solvent of water and ethanol How to do it is described.

  However, resveratrol has a low absorption rate and retention rate in the body and a short residence time in blood. For this reason, in recent years, a therapeutic agent that makes thrombosis prevention more effective by resveratrol having higher absorption rate and retention rate in the body and longer residence time in blood is required. Then, the present inventors discovered that the component extracted from a plant indo-quino can be used as a thrombosis preventive treatment, and came to this invention.

JP, 2012-41296, A

  The present invention provides a thromboprophylactic and therapeutic agent having a therapeutic effect higher than that of the prior art and a method for producing the same, by using an extract component obtained by alcohol extraction from the whole tree of Indian quinque (Pterocarpus marsupium).

The invention according to claim 1, the plant Indokinoki the (Pterocarpus marsupium) powdered relates thromboprophylaxis therapeutic agent comprising an extract obtained by intends row heating extraction with an alcohol solution from the powder.

Invention, the extraction obtained by heating extraction with alcohol solution, according to claim 1, characterized that it contains pterostilbene represented by the following formula (1) according to claim 2 Related to antithrombotic agents for thrombosis.

The invention according to claim 3 is an extraction obtained by: (1) pulverizing a plant indo-quinoki (Pterocarpus marsupium); and extracting an extract from the powder obtained in (2) (1) with an alcohol solution. The present invention relates to a method for producing an antithrombotic therapeutic agent containing a substance.
The invention according to claim 4, according to claim 3, characterized in that the extract obtained by the process of extracting the extract according to the alcoholic solution contains pterostiibene represented by the following formula (1) The present invention relates to a method for producing a thrombus preventing and treating agent.
The invention according to 請 Motomeko 5, the alcohol contained in the alcohol solution, methanol, ethanol, a manufacturing method of thromboprophylaxis therapeutic agent according to claim 3 or 4, characterized in that it is selected from the group consisting of propanol About.

According to the invention which concerns on Claim 1 and 3 , an extract effective as an antithrombotic therapeutic agent can be obtained from a plant indochiki through a process (1)-(2). Furthermore, since this extract can reduce the viscosity in blood and can suppress the occurrence of thrombus, it can provide an antithrombotic therapeutic agent effective for the treatment of thrombotic diseases .

According to the invention as set forth in claims 2 and 4 , since the extract obtained by performing heat extraction with an alcohol solution contains pterostilbene, a thrombus preventing and treating agent more effective for the treatment of thrombotic diseases is provided. Can be provided .

According to the fifth aspect of the present invention, by using methanol, ethanol and propanol as the alcohol solution, it is possible to extract an effective component for the antithrombotic agent from the indo-quinoqui.

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

The antithrombotic therapeutic agent according to the present invention can be obtained through the following steps.
(1) Extracting the extract with an alcohol solution from the powder obtained by the step (2) (1) of pulverizing the plant indo-quinoki (Pterocarpus marsupium) (3) ethyl acetate from the extracted powder obtained by the process (3) (2) Extracting an extract soluble in water

The method for producing the antithrombotic therapeutic agent according to the present invention will be described in detail.
First, step (1) will be described.
The plant indica is powdered by a crusher or the like.
Indo-kinokiki is Pterocarpus marsupium belonging to the leguminous genus Citan, and is an evergreen tree distributed from southern India to Sri Lanka. At this time, it is possible to use whole trees including stems (heartwood, sapwood, bark), branches, leaves, flowers, roots of indo-quinoki when powdered, or stems, branches, leaves, flowers, roots of indo-quinaki Or any combination thereof. Moreover, it does not specifically limit about the particle size of the powder obtained by pulverization.

The step (2) will be described.
Using a mixed solvent (alcohol solution) consisting of water and alcohol from powder of powdered indo-quinaki, components soluble in an alcohol solution are obtained. As the alcohol used at this time, methanol, ethanol, propanol or the like is suitably used. Furthermore, it is preferable that content of the alcohol contained in alcohol solution is 30 to 100%. If the alcohol content is less than 30%, it is not preferable because pterostilbene is difficult to extract. By using an alcohol solution for this operation, it is possible to extract an effective ingredient for treating thrombotic diseases contained in indo-quinaki. Furthermore, it is preferable that the mass ratio of the powdered indo-quinokia used for this extraction operation and an alcohol solution is 1: 5-20.

  The solution containing the component soluble in alcohol solution is concentrated to obtain a concentrate. By this operation, it is possible to increase the concentration of the component of the indo-quinaki dissolved in alcohol which is a component boiling lower than water. The concentration temperature in this operation is preferably 40 to 60 ° C. The reason is to prevent the decomposition of the components. Moreover, since there is a possibility that solids such as powder are contained in the extract after concentration, it is preferable to remove by filtration.

The step (3) will be described.
The concentrate obtained in the step (2) is dried under reduced pressure using a vacuum drier or the like to obtain an extraction powder (extract of solid). At this time, the apparatus used for vacuum drying is not particularly limited.

  Water and ethyl acetate are added to the obtained extracted powder to extract a component soluble in ethyl acetate from the extracted powder. After this, 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 multiple portions to extract a component (target extract) soluble in ethyl acetate contained in water.

  According to the above-described method, the obtained extract can reduce viscosity in blood and can suppress the occurrence of thrombus, and thus can be used as a thrombus preventing and treating agent effective for treating thrombotic diseases.

  The effects of the present invention are made more clear by showing examples regarding the antithrombotic therapeutic agent according to the present invention and the method for producing the same. However, the present invention is not limited to this embodiment.

<Production of antithrombotic agents>
Fifty grams of the whole tree (including stems, branches, leaves, flowers, and roots) of Indian quinoki was powdered using a grinder. 50 g of the obtained powder and 500 ml of a 50% ethanol solution in a distillation flask of a distillation apparatus (heat source (gas burner), distillation flask, double-sided tube, thermometer, condenser, cooling water, flask for storing distilled water) In addition, the temperature was raised to 90 ° C. to obtain a component which is soluble in a 50% ethanol solution from an indo-quinoki, and the solution was concentrated by an evaporator. Let this be an ethanol extract. The extract concentrated by vacuum distillation was dried under reduced pressure (40 ° C.) to obtain an extracted powder.
In a separatory funnel, 500 mg of the extracted powder, 50 ml of water and 40 ml of ethyl acetate were added to extract the components soluble in ethyl acetate. At this time, ethyl acetate was added in three 40 ml portions (total amount 120 ml). After this, concentration under reduced pressure gave an ethyl acetate extract (a thrombosis preventive and therapeutic agent). At this time, water added to the separatory funnel was used as a water extract.

<Component analysis>
The components of the extract powder and ethyl acetate extract in this example were analyzed by liquid chromatography.
The liquid chromatograph measuring device 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 x 250 mm)
Column temperature: 40 ° C
Mobile phase: A phase was a 30% methanol solution containing 0.1% phosphoric acid, and B phase was methanol.
30 minutes of liquid transfer so that A / B = (100/0) → A / B = (0/100).
Liquid transfer rate: 0.8 ml / min
Detection: UV (290 nm)
The results are shown in FIGS. 1 and 2.

<A test to improve blood rheology using endotoxin (LPS)-induced DIC diseased rats>
Male rats (8 weeks old, 7 rats in each group) preliminarily fed with each sample for 1 week were used.
(Control)
Controls in this study are healthy male rats. The rats were orally administered 0.2% sodium carboxymethylcellulose (CMC.Na) once a day for 7 days a day. After administration on day 7, blood was collected from the abdominal vena cava under pentobarbidal anesthesia.
(Carrier control)
The vehicle control in this study has LPS induced DIC pathology and does not receive components that are effective in preventing thrombus. Male rats were orally administered 0.2% sodium carboxymethylcellulose (CMC · Na) once a day for 7 days a day. One hour after subject administration on day 7, LPS was administered under mild anesthesia with pentobarbidal (a sleep medicine). Four hours after administration of LPS, blood was collected from the abdominal vena cava under pentobarbidal anesthesia.
(Positive control)
The positive controls in this study receive agents known to be effective in preventing thrombus. Male rats were administered heparin as a positive control drug one hour prior to LPS administration on day 7. Four hours after administration of LPS, blood was collected from the abdominal vena cava under pentobarbidal anesthesia.

Example 1
The subject prepared ethyl acetate extract at 50 [mg / kg] was orally administered once a day for 7 days on a daily basis. One hour after subject administration on day 7, LPS was administered under mild anesthesia with pentobarbidal (a sleep medicine). Four hours after administration of LPS, blood was collected from the abdominal vena cava under pentobarbidal anesthesia.
(Example 2)
The same as Example 1 except that a subject prepared by adjusting the ethyl acetate extract to 200 mg / kg was used.
(Example 3)
The same as Example 1 except that a subject prepared by adjusting the ethyl acetate extract to 500 [mg / kg] was used.

The blood collected was subjected to a biochemical examination for the number of platelets in blood, the number of white blood cells, the number of red blood cells, the amount of fibrinogen in serum, the amount of fibrin degradation product (FDP).
The whole blood passage time is determined by using the blood collected from the rat of each sample of the example, control, carrier control and positive control, and using the microchannel array Bloody 6-5 (width 4.5 μm, length 30 μm, depth 4) The measurement was carried out using a blood fluidity measurement apparatus (MC-FAN: manufactured by Hitachi, Ltd.) loaded with 5.
50 μL of one volume of 3.8% sodium citrate solution mixed with 9 volumes of each blood collected from rats of each sample of Example, Control, Carrier Control, and Positive Control is subjected to MC-FAN, 50 μL whole blood transit time was measured under 20 cm water pressure. The measurement value of the whole blood passage time was shown by converting the case where the passage time of the physiological saline was 12.0 seconds by the following equation, using the passage time of 100 μL of the physiological saline measured immediately before.
Whole blood transit time = blood transit time (s) × 12.0 (s) / saline transit time (s)
The results are shown in Tables 1 and 2.

<Platelet aggregation inhibition test>
(Preparation of PRP suspension)
Blood is 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 layer platelet rich plasma (PRP) was obtained. The obtained PRP was put into another silicon tube, then capped and stored at room temperature. Furthermore, the lower layer was centrifuged (1520 × g, 15 minutes, 25 ° C.) to obtain platelet-rich plasma (PPP) in the upper layer. PRP was diluted with PPP so that the platelet count would be 2.5-3.5 × 10 ⁵ / μL, and used as a PRP suspension. The PPP was set to a standard value of 100% transmission and the PRP suspension to a transmission of 0%.

(Preparation of test solution)
Positive Control The positive control used in the present test was a drug having an aggregation inhibitory effect. As a positive control drug, indomethacin is used, and indomethacin is dissolved in dimethyl sulfoxide (DMSO), and then 50 mM Tris-HCl buffer (Tris-HCl buffer, buffer adjusted to pH 7.4 with 1 M HCl solution) DMSO Dissolved at a concentration of 0.5% was used.

Negative control As a negative control in the present test, a substance which was effective for inhibiting aggregation was dissolved and dissolved in 50 mM Tris-HCl buffer so that the concentration of DMSO was 0.5%.

EXAMPLE An extract consisting of a component soluble in an ethanol solution from a powder of an indo-quinaki extract (ethanol extract), an extract consisting of a component soluble in ethyl acetate (ethyl acetate extract), and a component soluble in water Extract (water extract) Each was dissolved in dimethyl sulfoxide (DMSO), and then with 50 mM Tris-HCl buffer (Tris-HCl buffer, buffer adjusted to pH 7.4 with 1 M HCl solution) Dissolved at a concentration of 0.5% was used.

Comparative Example The same test was also conducted on pterostilbene and resveratrol which are conventionally known to have an aggregation inhibitory 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 suppression test)
200 μL each of the prepared PRP was dispensed in a cuvette dedicated to an aggregometer (PRP 313M, MC Medical Inc.) and stirred at 37 ° C. and 1000 rpm for 3 minutes, and then 11 μL of a test solution was added using a microsyringe. The mixture was further incubated and stirred at 37 ° C. and 1000 rpm for 3 minutes, and then 11 μL of collagen (100 μg / mL, dissolved in a diluted solution) was added as a coagulant by a microsyringe. The change in permeability of PRP suspension caused by platelet aggregation was recorded using an aggregometer for 10 minutes after addition of the aggregating agent. The 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 the PRP suspension and 100% for the PPP. The platelet aggregation rate of the subject was taken as the decreasing rate of the maximum permeability of the subject to the maximum permeability of the control group.
The results are shown in Tables 3 and 4.

<Results of preparation of antithrombotic agent>
In FIGS. 1 and 2, the vertical axis represents peak intensity, and the horizontal axis represents retention time. At this time, the peak indicated by retention time 28.14 [min] is a peak of pterostilbene.
As shown in FIG. 1, by using an ethanol solution as an extract, it was possible to extract an extract containing pterostilbene as a main component from a powder of an indo-quinaki. Further, as shown in FIG. 2, since the peak showing pterostilbene is higher than that in FIG. 1, the objective is to further fractionate the extract obtained by ethanol distillation from the extraction powder with ethyl acetate. An extract could be obtained selectively.

<Results of blood rheology improvement effect test>
As shown in the example of Table 1, it can be seen that the whole blood passage time is shortened as the target extract dose increases. From this result, it was found that the ethyl acetate extract (the target extract) extracted from the indo-quinaki with an alcohol solution has a function to reduce the viscosity of blood.

<Platelet aggregation inhibition test result>
As shown in Table 3, the extract extracted from the ethanol solution (ethanol extract) and the extract obtained from the component soluble in ethyl acetate (ethyl acetate extract) have a low platelet aggregation rate I understand.
Resveratrol and pterostilbene are known to be effective in reducing platelet aggregation. As shown in Table 4, although pterostilbene alone is effective, the ethyl acetate extract (target extract) extracted from indo-quinaki is more effective. This is considered to be due to the fact that the extract having an effect of reducing the aggregation rate is contained in the extract extracted from Indian quinque besides pterostilbene.

  The extract obtained by extracting an indo-quinaki by the antithrombotic agent and therapeutic agent according to the present invention and the method for producing the same is suitably used for an antithrombotic agent for thrombotic diseases.

Claims (5)

  An agent for preventing and treating thrombosis, which comprises an extract obtained by pulverizing a plant indica quinque (Pterocarpus marsupium) and subjecting the powder to heat extraction with an alcohol solution. The thrombosis preventive therapeutic agent according to claim 1, wherein pterostilbene represented by the following formula (1) is contained in an extract obtained by performing heat extraction with the alcohol solution.
(1) A thrombosis preventing and treating agent comprising an extract obtained by the steps of: (1) pulverizing a plant indoquinoki (Pterocarpus marsupium); and (2) extracting an extract with an alcohol solution from the powder obtained by (1). Production method. The method for producing a thrombus preventing and treating agent according to claim 3, wherein pterostilbene represented by the following formula (1) is contained in the extract obtained by the step of extracting the extract with the alcohol solution.
The method for producing a thrombus preventing and treating agent according to claim 3 or 4 , wherein the alcohol contained in the alcohol solution is selected from the group consisting of methanol, ethanol and propanol.