JP4877664B2 - HEALTH FOOD FOR PREVENTION OR IMPROVEMENT OF THROMBOSIS AND PHARMACEUTICAL COMPOSITION FOR PREVENTION OR TREATMENT OF THROMBOSIS - Google Patents

Description

  The present invention relates to a health food for preventing or improving thrombosis and a pharmaceutical composition for preventing or treating thrombosis.

In recent years, the pharmacological action of shiitake mushroom (Lentinus edodes) has attracted attention. For example, JP 2000-159683 discloses the action of shiitake on hepatic dysfunction, and JP 2003-155249 A Shiitake's IgA production promoting action is disclosed.
JP 2000-159683 A JP 2003-155249 A

However, the pharmacologically active ingredients of shiitake mushrooms so far are mainly water-soluble ingredients such as polysaccharides, and pharmacologically active ingredients other than shiitake polysaccharides, such as organic solvent extract components or organic solvents and water. The pharmacological action of the extracted components, particularly the essential oil components, from the mixture of these is not reported. In addition, the platelet aggregation inhibitory action of shiitake components has not been reported.
Among the components of shiitake mushrooms, the present inventors have proposed that an organic solvent extraction component or an extraction component based on a mixture of an organic solvent and water, particularly a component extracted into an organic solvent after an enzyme reaction has progressed for a certain period of time, inhibits platelet aggregation. The present invention was completed. The component includes the following formulas I, II, III, IV, V and / or VI:

The compound etc. which are represented by these are included. Among these compounds, the inventors of the present invention show that 1,2,3,5,6-pentathiepan (hereinafter referred to as lenthionine) represented by the above formula I has a high platelet aggregation inhibitory action. I found it.
Lentionine is known as an aroma component of shiitake mushroom, and the γ-glutamyl group of the precursor lentinic acid is eliminated by the action of γ-glutamyltransferase to produce desglutamyl lentinic acid, which has C-S lyase. By acting, sulfenic acid or thiosulfinate is generated, and a sulfur-containing fragment generated by non-enzymatic cleavage of thiosulfinate is generated by repeating condensation polymerization. At this time, in addition to lenthionine, sulfur-containing compounds represented by the above formulas II to VI are also produced.
Therefore, the present invention relates to an extract component of an organic solvent extract of shiitake mushroom or a mixture of an organic solvent and water (hereinafter sometimes referred to as “extract of shiitake mushroom of the present invention” or simply “extract of shiitake mushroom”), A compound of the above formula I, II, III, IV, V and / or VI which is an organic solvent-soluble component or a derivative thereof (hereinafter referred to as “an active ingredient of shiitake mushroom of the present invention” or simply “an active ingredient of shiitake mushroom”) The present invention relates to a health food for preventing or ameliorating thrombosis or a pharmaceutical composition for preventing or treating thrombosis.

1stly, this invention relates to the following health food.
(1) A health food for preventing or ameliorating thrombosis comprising an extract of an organic solvent of shiitake mushroom or a mixture of an organic solvent and water as an active ingredient.
(2) The health food according to (1), wherein the extract is an extract obtained from an organic solvent of a fruit body of shiitake mushroom or a mixture of an organic solvent and water.
(3) The health food according to (1), wherein the extract is an extract of shiitake fruiting bodies in an organic solvent.
(4) Any one of (1) to (3), wherein the extract is extracted after crushing shiitake mushrooms, preferably fruit bodies of shiitake mushrooms, and standing at a temperature of 3 to 70 ° C. for 1 minute or longer. Health food according to crab.
(5) A health food for preventing or ameliorating thrombosis comprising a compound represented by the following formulas I, II, III, IV, V and / or VI or a derivative thereof as an active ingredient.

(6) The health food according to (5), wherein the derivative is a compound represented by the following formulas VII, VIII, IX, X, XI and / or XII.

Wherein R ^{1 to} R ¹³ are a hydrogen atom, a hydroxyl group, a halogen atom, an unsubstituted or substituted alkyl group having 1 to 20 carbon atoms, an unsubstituted or substituted alkenyl group having 1 to 20 carbon atoms, An unsubstituted or substituted alkoxy group having 1 to 20 carbon atoms, an unsubstituted or substituted aryl group having 6 to 20 carbon atoms, and an unsubstituted or substituted alkyl aryl group having 7 to 20 carbon atoms)
(7) The health food according to (6), wherein the derivative is a compound represented by the formula VII.
(8) The health food according to (5), which contains, as an active ingredient, a compound represented by the formula I, II, III, IV, V and / or VI.
(9) The health food according to (5), which contains the compound represented by the formula I as an active ingredient.

Moreover, this invention relates to the following pharmaceutical composition.
(10) A pharmaceutical composition for preventing or treating thrombosis, which comprises, as an active ingredient, an extract of an organic solvent of shiitake mushroom or a mixture of an organic solvent and water.
(11) The pharmaceutical composition according to (10), wherein the extract is an extract obtained from an organic solvent of a fruit body of shiitake mushroom or a mixture of an organic solvent and water.
(12) The pharmaceutical composition according to (10), wherein the extract is an extract of shiitake fruiting body in an organic solvent.
(13) Any one of (10) to (12), wherein the extract is extracted after crushing shiitake mushrooms, preferably fruit bodies of shiitake mushrooms, and standing at a temperature of 3 to 70 ° C. for 1 minute or longer. A pharmaceutical composition according to claim 1.

(14) A pharmaceutical composition for preventing or treating thrombosis comprising the compound represented by the above formula I, II, III, IV, V and / or VI or a derivative thereof as an active ingredient.
(15) The pharmaceutical composition according to (14), wherein the derivative is a compound represented by the formula VII, VIII, IX, X, XI and / or XII.
Wherein R ^{1 to} R ¹³ are a hydrogen atom, a hydroxyl group, a halogen atom, an unsubstituted or substituted alkyl group having 1 to 20 carbon atoms, an unsubstituted or substituted alkenyl group having 1 to 20 carbon atoms, An unsubstituted or substituted alkoxy group having 1 to 20 carbon atoms, an unsubstituted or substituted aryl group having 6 to 20 carbon atoms, and an unsubstituted or substituted alkyl aryl group having 7 to 20 carbon atoms)
(16) The pharmaceutical composition according to (15), wherein the derivative is a compound represented by the formula VII.
(17) The pharmaceutical composition according to (14), which comprises a compound represented by the formula I, II, III, IV, V and / or VI as an active ingredient.
(18) The pharmaceutical composition according to (14), which contains the compound represented by the formula I as an active ingredient.
The present invention also relates to a method for preventing or treating thrombosis shown below.
(19) A method for preventing or treating thrombosis, comprising administering an extract of an organic solvent of shiitake mushroom or a mixture of an organic solvent and water.
(20) A method for preventing or treating thrombosis comprising administering a compound represented by the above formula I.
(21) A method for preventing or treating thrombosis, comprising administering the compound represented by the above formula I together with another antiplatelet drug.

According to the present invention, a health food effective for preventing or improving thrombosis and a pharmaceutical composition for preventing or treating thrombosis are provided. It is considered that the health food and pharmaceutical composition of the present invention have a platelet aggregation inhibitory effect with a mechanism of action different from cyclooxygenase inhibition. The active ingredient of shiitake mushrooms of the present invention, particularly lenthionine, does not participate in the platelet aggregation cascade caused by the signal transduction substance in platelets, and suppresses morphological changes such as platelet adhesion and release reaction, or the accompanying protein phosphorylation. This is considered to suppress platelet aggregation. Therefore, when used together with other antithrombotic agents, thrombosis can be more effectively prevented or treated by the effect of different action mechanisms.

It is a figure which shows the result of GC-MS analysis of the shiitake extract of this invention. It is a graph which shows the platelet aggregation inhibitory effect of a lenthionine. It is a graph which shows the platelet aggregation inhibitory effect of a lenthionine. It is a graph which shows the platelet aggregation inhibitory effect of the shiitake extract of this invention. It is a graph which shows the platelet aggregation inhibitory effect of the shiitake extract of this invention. It is a graph which shows the platelet aggregation inhibitory effect of a lenthionine. It is a figure which shows the action mechanism of platelet aggregation. It is a graph which shows the platelet aggregation inhibitory effect of a lenthionine, 1, 2- dithiepan, and 1, 2- dithiane. It is a graph which shows the platelet aggregation inhibitory effect of a lenthionine, 1, 2- dithiepan, and 1, 2- dithiane.

In the health food or pharmaceutical composition of the present invention, the raw shiitake may be raw shiitake or dried shiitake. The dried shiitake mushroom is produced by, for example, sun drying or mechanical drying using a drying furnace or the like. Shiitake may use either a fruit body or a mycelium, but preferably a fruit body.
The extraction solvent is an organic solvent or a mixture of an organic solvent and water, preferably an organic solvent. Examples of organic solvents include methanol, ethanol, n- or isopropanol, n-, iso, secondary or tertiary butanol, n-, iso, secondary or tertiary pentanol, n-, iso, secondary or secondary. Alcohols such as hexanol, ketones such as acetone and methyl ethyl ketone, esters such as methyl acetate, ethyl acetate and propyl acetate, halogenated hydrocarbons such as chloroform and methylene chloride, ethers such as diethyl ether, hexane, pentane, etc. Aliphatic hydrocarbons, aromatic hydrocarbons such as benzene, toluene and xylene, or a mixture thereof, preferably diethyl ether, but not limited thereto.
The extraction temperature is preferably 70 ° C. or lower, more preferably 40 ° C. or lower, for example, 5 to 30 ° C. (This is because the component to be extracted may decompose when heated at a temperature higher than 70 ° C. for a long time.)

The extraction method can be immersion in an organic solvent or a mixture of water and an organic solvent, digestion, heating under reflux cooling, and the like. Extraction may be carried out continuously or batchwise, for example, at a temperature ranging from room temperature to the boiling point of the solvent, under pressure, normal pressure or reduced pressure. The extraction time can be appropriately determined according to the extraction method, the extraction solvent, and the like. For example, in the case of extraction at 5 to 30 ° C., it is 1 minute to 24 hours, preferably 15 minutes to 30 minutes.
Prior to the extraction, pretreatment such as shiitake crushing and isothermal treatment is preferably performed. This is because the enzymatic reaction for producing a sulfur-containing compound such as lenthionine starting from lentinic acid contained in shiitake mushrooms is efficiently advanced.
The crushing is performed by crushing using a device such as a homogenator in a buffer solution having a pH of 3 to 12, for example.
The isothermal treatment is performed at a temperature of, for example, about 3 to 70 ° C., preferably 15 to 50 ° C., more preferably 25 to 40 ° C., for 1 minute to 24 hours, preferably 10 minutes to 12 hours, more preferably 30 minutes to 3 This is done by maintaining time.
The extract used in the health food or pharmaceutical composition of the present invention may be further purified by performing an extraction operation with an organic solvent, followed by filtration, drying, fractionation and the like. Filtration, drying and fractionation can be carried out by conventional methods well known to those skilled in the art.
Further, the extract used in the health food or pharmaceutical composition of the present invention may be extracted, purified if desired, concentrated, dried or freeze-dried, and further sterilized if desired. Good. Concentration or drying is carried out under normal pressure or reduced pressure, preferably at a temperature of 150 ° C. or lower, more preferably at 30 ° C. or lower. It is because the component which should be extracted may decompose | disassemble when it exceeds 150 degreeC.
However, the health food and pharmaceutical composition of the present invention are not limited to those containing the extract obtained by the above-described extraction / purification method, and may contain any essential oil components of shiitake mushrooms such as lenthionine. .

The health food or pharmaceutical composition of the present invention may contain a compound represented by the above formulas I to VI as an active ingredient. These compounds may be contained as components in the extract, or may be compounds obtained by synthesis.
The compound represented by the above formula I (lenthionine) is prepared by the method described in, for example, Journal of the Chemical Society, Perkin Transactions 1: Organic and Bio-Organic Cemistry (1972-1999), (3), 509-514; 1990. Can be synthesized.
The process consists of reacting diiodomethane and disodium disulphide Na ₂ S ₂ in a water: dichloromethane bilayer mixture at room temperature.
Specifically, it is synthesized by the following method. A solution of disodium disulphide (2.8 g, 25.5 mmol) in water (100 cm ³ ) is added to a solution of diiodomethane (6.81 g, 25.5 mmol) in methylene chloride (100 cm ³ ). The bilayer mixture is stirred vigorously at room temperature for 5 days. The pale yellow aqueous layer becomes colorless. The organic layer is separated and dried over sodium sulfate. Chromatography first elutes methaneiodide, and then 1,2,3,5,6-pentathiepan (melting point 61 ° C.) is obtained in a yield of about 18%.

In addition, lenthionine can be produced by the method described in, for example, Tetrahedron Vol. 41, No. 22, pp. 5145-5158.
The method consists of reacting a metal halide with a metal tetrathiopercarbonate.
Specifically, it is synthesized by the following method.
Sodium tetrathiopercarbonate (0.10 mol) in 40 ml of ethanol was treated with ether to precipitate the crystalline salt. Add 27.3 g (0.10 mol) of methylene iodide in 100 ml of ethanol with stirring, and when all the precipitate has dissolved, add 750 ml of water. The ether layer is separated, washed 3 times with water, dried over calcium chloride and evaporated to give 13.7 g of a yellow oil. Purification by column chromatography on silica gel and further dissolution of the resulting crude product in acetone and precipitation at −25 ° C. yields lenthionine (melting point 58-61 ° C.) in 25% yield.

Compounds represented by the above formula II are described in, for example, Phosphorus and Sulfur and the Related Elements, 15 (1), 27-32; 1983, Phosphorus and Sulfur and the Related Elements, 8 (2), 157-9; 1980, etc. It can be synthesized by the method described.
Moreover, the health food or pharmaceutical composition of the present invention may contain a derivative of the compound represented by the formula I, II, III, IV, V and / or VI as an active ingredient. Such derivatives are, for example, compounds represented by the above formulas VII, VIII, IX, X, XI and / or XII.
This can be produced by introducing a substituent into the compound of the above formula I, II, III, IV, V and / or VI isolated and purified as described above or synthesized as described above by a conventional method.
Since the extract of shiitake mushroom or the active ingredient of shiitake mushroom of the present invention has high activity and low toxicity, it can be used safely in medicine.

(healthy food)
The “health food” of the present invention is an extract (soft extract, dry extract), capsule, granule, powder, tablet, liquid preparation containing the above-described extract of shiitake mushroom or the active ingredient of shiitake mushroom as an active ingredient. , Soaking agent, decoction, troche, flow extract, tincture and the like. These can be formulated by methods known in the art. Health foods in the form of these formulations may contain the adjuvants listed below for the pharmaceutical composition. The compounding amount of the shiitake extract or shiitake active ingredient of the present invention varies depending on the dosage form. The active ingredient dose per day is, for example, 1 to 15 g, preferably 2 to 5 g as the lyophilized weight of the organic solvent extract component, and for example, 0.1 to 1.5 g, preferably in the case of a compound or a derivative thereof. Is an amount of 0.2 to 0.5 g.

The health food of the present invention may be a food obtained by adding the active ingredient of shiitake mushroom of the present invention to a food such as a general processed food, or a so-called functional food. Examples of such foods include rice cakes, gums, jelly, biscuits, cookies, rice crackers, breads, noodles, fish and livestock meat products, tea, soft drinks, coffee drinks, and dairy drinks to which the active ingredient of the present invention is added. , Whey beverage, lactic acid bacteria beverage, yogurt, ice cream, pudding and the like. Furthermore, the health food of the present invention also includes health liquor in which the shiitake mushroom and optionally other ingredients are soaked in drinking alcohol.
In the case of such a functional food, it is produced by the usual method for producing the processed food, except that the required amount of the extract of shiitake of the present invention or the active ingredient of shiitake is added to each food material. be able to. In this case, the amount of the shiitake extract of the present invention or the active ingredient of shiitake varies depending on the food to be added. For example, the lyophilized weight of the organic solvent extract component of shiitake is, for example, 0.001 to 10% by weight, preferably Is 0.1 to 1% by weight. Moreover, when mix | blending as a compound or its derivative (s), it is 0.0001 to 10 weight%, for example, Preferably it is 0.01 to 0.1 weight%.

(Pharmaceutical composition)
The “pharmaceutical composition” of the present invention contains the active ingredient of shiitake mushroom of the present invention as an active ingredient. The dosage form is not particularly limited, but for example, extract (soft extract, dry extract), capsule, granule, solution, emulsion, suspension, powder, tablet, liquid, immersion agent, decoction, troche, It may be a flow extract, tincture, eye drops, nasal solution, ointment, cream, lotion, injection, suppository and the like. The amount of the extract of shiitake mushroom or the active ingredient in the pharmaceutical composition of the present invention varies depending on the dosage form. Moreover, the active ingredient dosage per day is 1-15 g, for example, as a freeze-dried weight of an organic-solvent extraction component, Preferably it is 2-5 g. Moreover, when mix | blending as a compound or its derivative (s), it is 0.1-1.5g, for example, Preferably it is 0.2-0.5g.

These preparations are pharmaceutical preparation techniques such as excipients, binders, disintegrants, lubricants, stabilizers, flavoring agents, solubilizers, suspension agents, coating agents, diluents etc. It is formulated with known auxiliaries that can usually be used in the field.
The pharmaceutical composition containing the active ingredient of shiitake mushroom of the present invention is usually prepared according to a conventional method and formulated in a form suitable for the purpose. The pharmaceutical composition may contain an active ingredient of the shiitake mushroom of the present invention and adjuvants known to be usable in the pharmaceutical formulation art.

  Solid preparations are, for example, powders, granules, tablets, capsules, sugar-coated tablets and the like. The compound of the present invention as an active ingredient and a diluent (for example, lactose, dextrose, sucrose, cellulose, corn starch, potato starch, etc. ), Lubricants (eg silica, talc, stearic acid, magnesium stearate, calcium stearate, polyethylene glycol, etc.), binders (eg starches, gum arabic, gelatin, methylcellulose, carboxymethylcellulose, polyvinylpyrrolidone etc.), discrete agents (For example, starch, alginic acid, alginate, etc.), a saturant, a coloring agent, a sweetener, a wetting agent (for example, lecithin, polysorbate, lauryl sulfate, etc.) and the like can be contained. These can be formulated by known methods such as mixing, granulating, tableting, sugar coating and the like.

Liquid preparations can be in the form of, for example, syrups, solutions, emulsions and suspensions.
The suspension and emulsion can contain, for example, natural rubber, agar, sodium alginate, pectin, methylcellulose, carboxymethylcellulose, polyvinyl alcohol and the like as a carrier.
Intramuscular suspensions or solutions contain, as a pharmacologically acceptable carrier, for example, sterile water, olive oil, ethyl oleate, glycols such as propylene glycol, and as appropriate an appropriate amount of lidocaine hydrochloride can do. Intravenous injection or infusion solutions can contain, for example, sterile water as a carrier, but are preferably in the form of sterile isotonic saline solution.
The health food and pharmaceutical composition of the present invention are used for the prevention, amelioration or treatment of diseases caused by platelet aggregation such as cerebral infarction and myocardial infarction.
The present invention also relates to a method for preventing or treating thrombosis, which comprises administering the shiitake extract component or shiitake mushroom active component of the present invention described above together with other antiplatelet agents as desired.
Examples of other antiplatelet agents include ticlopidine hydrochloride, cilostazol, ethyl icosapentate, beraprost sodium, sarpogrelate hydrochloride, aspirin / dialuminate combination agent, aspirin, etc. It can be administered in combination with one or more drugs.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these do not limit the present invention.
Example 1: Preparation of shiitake extract 500 ml of 0.2 M Tris-HCl buffer (pH 3.0, 5.0, 7.0 or 9.0) was added to 300 g of fresh shiitake fruiting body, and homogenizer (manufactured by Matsubara). For 3 minutes. Then, it incubated at 37 degreeC for 30 minutes.
To this, 50 g of celite was added and filtered. The filtrate and the residue were extracted 5 times with 150 ml of methylene chloride, and the combined extracts were dried over sodium sulfate and filtered. The filtrate was concentrated and lipids were removed by passing through silica gel column chromatography.
The resulting oily substance was analyzed by GC-MS. The components obtained when incubated at pH 3.0, 5.0, 7.0 and 9.0 are shown in Table 1 below.

Example 2: Preparation of shiitake extract 500 ml of 0.2 M Tris-HCl buffer (pH 7.0) was added to 300 g of fruit bodies of fresh shiitake mushrooms, and homogenized with a homogenizer (manufactured by Matsubara) for 3 minutes. Then, it incubated at 37 degreeC for 30 minutes.
To this, 50 g of celite was added and filtered. The filtrate was distilled at 100 ° C. under normal pressure using an LN distillation apparatus (manufactured by Tanaka Rika Glass Co., Ltd.). 80 ml of methylene chloride was added to the obtained fraction. The organic phase was separated, dried over sodium sulfate, filtered and the filtrate was concentrated.

Example 3: Preparation of shiitake extract A shiitake extract was prepared in the same manner as in Example 2 except that incubation at 37 ° C for 30 minutes was not performed.

Example 4 Preparation of Shiitake Mushroom Extract 50 g of dried shiitake fruiting bodies were immersed in 900 ml of 0.2 M Tris-HCl buffer (pH 9.0) at 4 ° C. for 30 minutes, and then 3 with a homogenizer (manufactured by Matsubara). Homogenized for minutes and then incubated at 37 ° C. for 3 hours.
To this, 50 g of celite was added and filtered. The filtrate and the residue were extracted 5 times with 150 ml of methylene chloride, and the combined extracts were dried over sodium sulfate and filtered. The filtrate was concentrated and lipids were removed by passing through silica gel column chromatography.
The resulting oily substance was analyzed by GC-MS. The chart is shown in FIG. The obtained components are shown in Table 1 above.

Test Examples The following reagents and instruments were used in the following test examples.
Dulbecco's PBS (-) solution: A solution obtained by dissolving 9.6 g of Dulbecco's PBS (-) manufactured by Nissui Pharmaceutical Co., Ltd. in distilled water to a total volume of 1000 ml was used.
Instruments: Glass products (such as cuvettes) and metal instruments (such as steering rods) used for measurement are all made of silicon (SIGMACOTE, manufactured by Sigma-Aldrich Japan) to prevent adsorption and coagulation caused by platelet foreign body contact stimulation. What gave the coating process was used.

Test Example 1: Inhibitory effect of lenthionine on platelet aggregation by arachidonic acid Lentionine (manufactured by Ogawa Fragrance Co., Ltd.) was dissolved in ethanol containing 50 ppm of L-ascorbyl palmitate as an antioxidant, and each final concentration was 3.16 × 10. ^{-4, 1.0 × 10 -4, 3.16} × 10 -5, 1.0 × 10 -5, 3.16 × 10 -6, 1.0 × 10 -6, 3.16 × 10 -7 A diluted lenthionine solution was prepared by diluting to 1.0 × 10 ⁻⁷ .
Dissolve 100 mg of arachidonic acid (manufactured by Sigma-Aldrich Japan) in 2.76 ml of 0.5 N NaOH, dispense 50 μl each into an Eppendorf tube, add nitrogen gas, store frozen at −80 ° C., and use in PBS buffer A solution diluted to a concentration of 1.5 mM in PRP in the solution was used as the aggregation inducer solution.

Blood was collected from healthy adults (21 to 23 years old) who had not taken medicine for 2 weeks or more using a vacuum blood collection tube so that the volume was 9 parts by volume of blood and 1 part by volume of 3.8% sodium citrate.
The obtained citrated blood was centrifuged at 1500 rpm for 10 minutes, and the upper layer was used as platelet rich plasma (hereinafter referred to as PRP). The lower layer was further centrifuged at 3000 rpm for 15 minutes, and the upper layer was used as platelet poor plasma (hereinafter referred to as PPP).
300 μl of the PPP prepared above was dispensed into a cuvette, and 300 μl of the PRP prepared above was dispensed into another cuvette. This was set in an aggregometer (NKK HEMA TRACER 1 MODEL PAT-4A, SSR ENGINEERING), and the stirrer was rotated at 1000 rpm and incubated at 37 ° C.

The transmittance was recorded by a recorder (CHROMAPAC C-R4A, manufactured by Shimadzu Corporation). First, SPAN adjustment was performed by setting a cuvette containing PPP and adjusting the permeability of the recorder to 40000, and then setting a cuvette containing PRP and adjusting the permeability of the recorder to 0. This SPAN adjustment was performed for each measurement.
Next, after setting the cuvette containing the above PRP and starting the recorder, 2 μl of each concentration of lenthionine diluted solution prepared above was added, and after 3 minutes, 18 μl of the aggregation inducer solution prepared above was added. The change in aggregation curve was recorded for 10 minutes upon addition.
As a control, only the dilution solvent was added instead of the lenthionine dilution, and the PRP permeability was measured by the same method as described above.

The platelet aggregation inhibition rate was determined as follows.
The change in PRP permeability (T%) associated with platelet aggregation was regarded as the change in platelet aggregation rate (Agg%), and the maximum value was defined as the maximum aggregation rate (Agg.max%). The maximum agglomeration ratio (T and T ′, respectively) determined for the PRP to which the sample was not added and the PRP to which the sample was added was calculated, and the inhibition rate was calculated from the following formula using this value.

Inhibition rate (%) = (1−T ′ / T) × 100

The results are shown in the graph of FIG. In the graph, the concentration of the sample is shown on the horizontal axis, and the inhibition rate at the concentration is shown on the vertical axis. From this graph, the concentration (M) that inhibits aggregation by 50%, that is, the IC ₅₀ value, was 1.62 × 10 ⁻⁴ M.

Test Example 2: Inhibitory effect on platelet aggregation by U-46619 of lenthionine As an aggregation inducer solution, 1 mg of U-46619 (U-46619 1 mg / methyl acetate 100 μl, manufactured by Funakoshi Co., Ltd.) instead of the arachidonic acid solution) Except that a 10 mM U-46619 solution frozen at −20 ° C. was diluted by diluting with 185 μl of ethanol and diluted to a final concentration of 1 μM in PRP with PBS buffer at the time of use. In the same manner as in Test Example 1, the inhibitory action of lenthionine on platelet aggregation was examined.
The results are shown in the graph of FIG. In the graph, the concentration of the sample is shown on the horizontal axis, and the inhibition rate at the concentration is shown on the vertical axis. From this graph, the IC ₅₀ value was determined to be 1.97 × 10 ⁻⁴ M.

  Aspirin is known as a compound having an inhibitory action on platelet aggregation. The inhibitory action of aspirin on platelet aggregation is due to cyclooxygenase inhibition, thereby preventing the production of thromboxane A2 having a strong platelet aggregation action. In addition, the production of prostacyclin having a platelet aggregation inhibitory action produced by the same mechanism is also inhibited. However, according to this test example, when U-46619, which is a similar compound of thromboxane A2, was used as an inducing substance (since thromboxane A2 is an unstable compound), it also showed an inhibitory action on platelet aggregation. Accordingly, it is considered that the platelet aggregation inhibitory action of lenthionine is due to an action mechanism different from that of cyclooxygenase inhibition. Therefore, it is considered that the present invention can solve the problems when the aspirin is used as a platelet aggregation inhibitor.

Test Example 3: Inhibition of platelet aggregation by arachidonic acid of shiitake mushroom organic solvent extract The above test example except that the mushroom organic solvent extract prepared in Example 4 was used as a sample instead of the lenthionine solution. By the same method as 1, the inhibitory effect on platelet aggregation by arachidonic acid was examined.
The results are shown in the graph of FIG. In the graph, the concentration of the sample is shown on the horizontal axis, and the inhibition rate at the concentration is shown on the vertical axis. From this graph, the concentration (M) at which aggregation was inhibited by 50%, that is, the IC ₅₀ value, was found to be 201.3 μg / ml.
Thereby, it is clear that the organic solvent extract of shiitake has a high platelet aggregation inhibitory action.
Test Example 4: Inhibitory effect of Shiitake mushroom organic solvent extract on platelet aggregation by U-46619 The above test, except that the mushroom organic solvent extract prepared in Example 4 was used instead of the lenthionine solution as a sample. In the same manner as in Example 2, the inhibitory effect on platelet aggregation by U-46619 was examined.
The results are shown in the graph of FIG. In the graph, the concentration of the sample is shown on the horizontal axis, and the inhibition rate at the concentration is shown on the vertical axis. From this graph, the concentration (M) that inhibits aggregation by 50%, that is, the IC ₅₀ value, was found to be 549.4 μg / ml.
Thereby, it is clear that the organic solvent extract of shiitake has a high platelet aggregation inhibitory action.

Test Example 5:
The platelet aggregation inhibitory action of lenthionine when A23187, phorbol 12-myristate 13-acetate (hereinafter referred to as PMA), platelet activating factor (hereinafter referred to as PAF), and ADP were used as the inducing substances was examined.
As the lenthionine dilution, the lenthionine dilution prepared in Test Example 1 was used.
A solution of each inducer was prepared as follows.
A23187: A23187 manufactured by Wako Pure Chemical Industries, Ltd. was dissolved in DMSO and stored refrigerated as an A23187 solution having a final concentration of 12.5 mM. At the time of use, it was diluted with PBS buffer so that the final concentration in PRP was 50 μM.
PMA: PMA manufactured by Funakoshi Co., Ltd. was dissolved in ethanol and stored refrigerated as a PMA solution having a final concentration of 90 μM. At the time of use, it was diluted with 4% ethanol-containing PBS buffer so that the final concentration in PRP was 5 μM.
PAF: 10 mg of PAF manufactured by Funakoshi Co., Ltd. was dissolved in chloroform and stored frozen at −20 ° C. as a 1 mM PAF solution. At the time of use, the amount used is dispensed into an Eppendorf tube, chloroform is removed with nitrogen gas, PBS buffer containing 0.25% BSA is added, and sonication is performed. The final concentration in PRP is 1 μM. Diluted to
ADP: ADP manufactured by Wako Pure Chemical Industries, Ltd. was diluted with PBS buffer so that the final concentration in PRP was 10 μM.

Blood was collected from healthy adults (21 to 25 years old) who had not taken medicine for 2 weeks or more using a vacuum blood collection tube so that 9 parts by volume of blood was 1 part by volume of 3.8% sodium citrate.
Since blood is prone to spontaneous aggregation due to cooling, the following operations were performed at room temperature. The citrated blood obtained by blood collection was centrifuged at 1500 rpm for 10 minutes, and the upper layer was used as platelet rich plasma (hereinafter referred to as PRP). The lower layer was further centrifuged at 3000 rpm for 15 minutes, and the upper layer was used as platelet poor plasma (hereinafter referred to as PPP). Platelets were used within 3 hours after blood collection because platelet function declined about 3 hours after blood collection.
Platelet aggregation ability measurement When a platelet aggregation-inducing substance is added to PRP and stirred, platelets rapidly form aggregates, and the permeability of PRP increases as platelet aggregates are formed. The platelet aggregation ability was measured using this phenomenon.
Platelet aggregation ability was measured using an aggregometer (NKK HEMA TRACER 1 MODEL PAT-4A, SSR ENGINEERING). The cuvette was incubated at 37 ° C., and the rotation speed of the stirrer was set to 1000 rpm.
The transmittance was recorded by a recorder (CHROMAPAC C-R4A, manufactured by Shimadzu Corporation). First, a cuvette with a steering rod was prepared, 300 μl of PPP was placed in one cuvette, and 300 μl of PRP was dispensed in the other cuvette. Next, SPAN adjustment was performed by setting a cuvette containing PPP and setting the permeability of the recorder to 40000, and then setting a cuvette containing PRP and setting the permeability of the recorder to 0. . This SPAN adjustment was performed for each measurement.
Next, after setting the cuvette containing the above PRP and starting the recorder, 2 μl of each concentration of lenthionine diluted solution prepared above was added, and after 3 minutes, 18 μl of the aggregation inducer solution prepared above was added. The change in aggregation curve was recorded for 10 minutes upon addition.
As a control, a dilution solvent alone was used instead of the lenthionine dilution.
The platelet aggregation inhibition rate was determined by the same method as in Test Example 1.
The results are shown in the graph of FIG.
As apparent from FIG. 6, A23187 which causes platelet aggregation by increasing intracellular Ca ion concentration, PMA which causes platelet aggregation by activating protein kinase C, PAF which is a platelet activator, platelet aggregation Accordingly, lenthionine showed an inhibitory effect on platelet aggregation caused by any inducer of ADP that is released from the darkly stained granules in platelets and acts on other platelets. The action mechanism of each inducer is summarized in FIG. As is clear from this, lenthionine does not participate in the platelet aggregation cascade caused by the signaling substance in platelets, and suppresses morphological changes such as platelet adhesion and release reaction, or the accompanying protein phosphorylation. Conceivable.

Test Example 6:
In order to investigate which part of the structure the inhibitory action of lentionine on platelet aggregation is derived, we synthesized cyclic sulfur compounds similar in structure to lenthionine and measured their respective platelet aggregation inhibitory activities. The correlation was examined.
First, focusing on the disulfide structure and the cyclic structure of lenthionine, 1,2-dithiepan and 1,2-dithiane, which are cyclic sulfur compounds having a disulfide structure, were synthesized and compared with the action of lenthionine.
Synthesis of 1,2-dithiepan 4.02 mol (547.84 g) of 1,5-pentanedithiol was dissolved in 5 ml of dichloromethane and added to 25 ml of dichloromethane to which 5 g of silica gel hydrated with 2.5 ml of pure water was added. In addition, 4.06 mmol (208.97 mg) of bromine dissolved in 3.5 ml of dichloromethane was added dropwise with stirring. After completion of dropping, the mixture was stirred for 10 minutes, filtered through filter paper, concentrated by a rotary evaporator, and subjected to silica gel column chromatography (mobile phase: dichloromethane: hexane = 1: 2) to obtain 1,2-dithiepan.
Synthesis of 1,2-dithiane 4.02 mol (491.45 mg) of 1,4-butanedithiol was dissolved in 5 ml of dichloromethane, and 5 g of silica gel hydrated with 2.5 ml of pure water was added to 25 ml of dichloromethane. In addition, 4.06 mmol (208.97 mg) of bromine dissolved in 3.5 ml of dichloromethane was added dropwise with stirring. After completion of dropping, the mixture was stirred for 10 minutes, filtered through a filter paper, concentrated by a rotary evaporator, and subjected to silica gel column chromatography (mobile phase: dichloromethane: hexane = 1: 2) to obtain 1,2-dithiane.
The 1,2-dithiepan and 1,2-dithiane synthesized above were diluted at the same dilution rate as the diluted solution of lenthionine, and the inhibitory action of platelet aggregation by arachidonic acid was examined in the same manner as in Test Example 1, and Test Example 1 IC ₅₀ value was determined by the same method as above. The results are shown in FIG. As Figure apparent from, _{IC 50} of lenthionine are 1,2 dithiepane lower than _{IC 50} of 1,2-dithiane, platelet aggregation inhibitory action of lenthionine is 1,2 dithiepane, 1,2-dithiane of It turns out that it is higher than an aggregation inhibitory effect.

Test Example 7:
The platelet aggregation inhibitory effect on various inducers of lenthionine, 1,2-dithiepan and 1,2-dithiane at a concentration that inhibits arachidonic acid aggregation by 100% was examined by the same method as in Test Example 5. The results are shown in the graph of FIG. As is clear from the graph, lenthionine suppresses platelet aggregation caused by any initiator, but 1,2-dithiepan and 1,2-dithiane have a low inhibitory effect on platelet aggregation by inducers other than arachidonic acid. There was almost no inhibitory effect on A23817 or PMA. This indicates that the inhibition of platelet aggregation by lenthionine is not limited to the disulfide structure.

  According to the present invention, a health food effective for preventing or improving thrombosis and a pharmaceutical composition for preventing or treating thrombosis are provided.

Claims (7)

A pharmaceutical composition for preventing or treating thrombosis comprising an extract of shiitake mushrooms as an active ingredient. A pharmaceutical composition for the prevention or treatment of thrombosis, which comprises, as an active ingredient, an extract of shiitake mushroom that contains lenthionine as an extract component. The pharmaceutical composition for preventing or treating thrombosis according to claim 1 or 2, wherein the organic solvent is methylene chloride or ethanol. The pharmaceutical composition for preventing or treating thrombosis according to claim 1 or 2, wherein the organic solvent is methylene chloride. The pharmaceutical composition according to any one of claims 1 to 4 , wherein the extract is an extract of a fruit body of shiitake mushroom. The pharmaceutical composition according to any one of claims 1 to 5 , wherein the extract is extracted after crushing shiitake mushrooms and allowed to stand at a temperature of 3 to 70 ° C for 1 minute or more. A pharmaceutical composition for preventing or treating thrombosis comprising lenthionine as an active ingredient.

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