FR2709965A1 - Pharmaceutical compositions having antithrombotic activity and process for their preparation - Google Patents

Abstract

The present invention relates to a pharmaceutical composition having an antithrombotic action, comprising, as active component, at least one green tea extract and at least one pharmaceutically acceptable adjuvant. A subject of the invention is also a process for the preparation of an extract of dried green tea leaves, of dried green tea or of soluble tea, as well as the use of this extract for the manufacture of a pharmaceutical composition having a anti-thrombotic activity.

Description

Pharmaceutical compositions having antithrombotic activity and

  The present invention relates to a pharmaceutical composition having antithrombotic activity, inhibiting thrombogenesis and inhibiting

  of the cause causing thrombogenesis.

  In particular, the subject of the present invention is a pharmaceutical composition having antithrombotic, thrombogenesis inhibitory and thrombogenesis-inducing cause, containing as an active ingredient a pharmacologically acceptable extract of green tea leaves. Said extract is novel, stable and exceptionally inhibits the cause causing thrombogenesis, and is prepared

according to an improved method.

  The terms "antithrombotic", "thrombogenesis inhibitor" and "thrombogenesis causing cause inhibitor" will be referred to below.

under the term "antithrombotic".

  Green tea is used in the Far East, starting with China and

  Japan, as a luxury product for the table having medicinal virtues.

  Green tea leaves contain tannins, caffeine, ascorbic acid, tocopherols, flavonols, polysaccharides and 3-carotene, etc., as chemical constituents. Among them, caffeine, ascorbic acid and green tea tannins are the main constituents. Caffeine

  and ascorbic acid are contained in many other kinds of tea.

  However, green tea tannins represented by catechins are unique constituents found only in green tea. The four main catechins of green tea are epicatechin, epicatechin gallate, epigallocatechin and epigallocatechin gallate. Other inorganic substances such as fluorine, zinc, calcium and magnesium are

  also contained in green tea.

  It has already been known that green tea catechins have varied effects, including, as a main effect, the inhibition of lipid hyperoxidation and, additionally, the inhibition of the reaction of active oxygen which is supposed to cause many diseases, inhibition of cholesterol reabsorption, anticarcinogenic activity, antiviral-antibacterial activity, anticaries activity and deodorant activity, etc. In the early 1990s, it was discovered and described that epigallocatechin gallate, one of the catechins, inhibits the formation of carcinogenic compounds and the action of carcinogenic promoters. Consequently, given the encouraging results, it was expected that catechins from green tea

anticancer drugs.

  Important research by the present inventors on the pharmacological effects of green tea components has surprisingly and unexpectedly found that these green tea components

  exhibit exceptional antithrombotic activity.

  The present invention therefore relates to a pharmaceutical composition having antithrombotic activity, said composition comprising at least one green tea extract as active component and at least one pharmaceutically acceptable adjuvant. Among the pharmaceutically acceptable adjuvants, there may be mentioned vehicles, diluents and antioxidants. Catechins are isolated from green tea, according to a process that can

  be divided into three stages: 1) extraction, 2) elimination and 3) purification.

  The extraction is carried out in a known manner with hot water, cold water, methanol, ethanol or a mixture thereof in suitable proportions. To remove components other than the active components, strongly polar solvents such as chloroform, ethyl acetate or diethyl ether, which are organic solvents which do not mix with water, are used.

  in order to optimize the disposal operation.

  For the final purification, the extraction-sharing process is used, of which

  the mechanism is based on the polarity difference of the organic solvents.

  Currently, the methods of column chromatography and high-speed liquid chromatography are used for purification. The purification method by a pulling extraction method poses some problems such as the delay in establishing the equilibrium, the high cost of maintenance and the risks accompanying the use of organic solvents such as chloroform, methyl isobutyl ketone, etc. For the chromatographic method, sephadex or polymers are commonly used, but this method of purification still needs to be improved to the extent that the extraction conditions are complex, last a long time and require a lot of manpower. On the other hand, although purification by high speed liquid chromatography can save time and be more efficient, it is however unsatisfactory insofar as the system and the eluents are expensive, and the separation column is expensive.

can bask easily.

  To eliminate the problems described above, the present inventors have conducted extensive research on various methods. The present inventors have thus been able to remove the residue from the hydrothermal extraction by centrifugation, and by using a Sephadex LH-20 chromatography column and a simpler separation solvent, they have succeeded in eliminating the terpenoid and coloring components as well as the water-soluble caffeine, free amino acids and saccharide by rinsing with a 15% water-acetone or dioxane mixed solution. Accordingly, the further caffeine removal step provided in the known process is not necessary in the process according to the present invention. In addition, it is thus possible effectively to avoid the introduction of contaminants by the organic solvents or to eliminate the contaminants found in the final product as a result of the use of chloroform, methyl isobutyl ketone, etc. As a result, only the yellow-red portion is placed in the upper layer of the chromatography column and can be easily observed with the naked eye. On the other hand, the active component of the rest of the yellow-red part can be obtained in a maximum yield, by elution with a mixed solvent water-acetone or dioxane at%, all at once. The method according to the present invention is free from the disadvantages of known methods. The present invention also relates to a process for preparing a green tea extract comprising the steps of hydrothermally extracting dried green tea leaves, dried green tea or soluble tea with water. or with a solution of water and a low molecular weight alcohol to remove insoluble substances, concentrate the resulting extract, redissolve the resulting concentrate with water, extrate the resulting solution with a solvent organic to remove non-polar substances, concentrate the resulting aqueous layer, and reconcentrate the liquid separated from the concentrate by a separation method on

column.

  In the first step of a particular embodiment of the process of the present invention, green tea is extracted with hot water. Preferably, about 1/4 of n-butanol is then added to the extract, to protect the active components from oxidation, and then, while maintaining the quantity of n-butanol, the concentrate is concentrated throughout the duration of the operation. way to avoid the oxidation of the active compounds by the air on the surface. The final concentration of the active components can be achieved by lyophilization, thus completely eliminating the water, while maintaining a final ratio of

  concentrated to substantially constant active components.

  The dry powder of the active components thus obtained is yellow-brown, very soluble in water and stable for more than 6 months, not being hygroscopic. The dry powder can thus be easily transformed into

pharmaceutical product.

  It has further been found, after repeated tests of the process mentioned above, that each batch contains the active components in a ratio of

constant composition.

  The composition of the active fraction is as follows: 30% of polyphenols, 60% of a mixture of epigallocatechin gallate and

  epicatechin, and only 6% epigallocatechin.

  A particular method of extracting green tea extracts according to the present invention will now be described in detail. Extraction In the first step, dried leaves of green tea or dried tea are subjected as starting material of extraction, for 10 minutes with hot water at 70-90 ° C. or with a hydroalcoholic solution. Preferably, the extraction is completed by stirring, without allowing the boiling to occur. The insoluble materials of the extract are treated by a known method such as pressure filtration, vacuum filtration or centrifugal filtration. The amount of hot water used for the extraction may be from 5 to 150 times the weight of green tea, and preferably from 10 to 30 times the weight of green tea. The proportion of extraction solvent in hot water is for example -50% alcoholic solvent. Although the extraction time depends on the temperature, it is generally from 30 seconds to one hour, and preferably,

from 5 minutes to 20 minutes.

  Concentration under reduced pressure When hot water is used as the extraction solvent, 10-50% (v / v) of n-butanol, preferably 20%, is added in one embodiment according to one embodiment. extract obtained, and this proportion is maintained until the end of the concentration step under reduced pressure, in order to avoid the boiling of the extract, and to prevent the direct contact of the active components contained in the extract with the air, by a layer of n-butanol covering the extract, which avoids

  a modification of the content of active components of the composition.

  The concentration is carried out under reduced pressure, heating to 70 mm Hg, for example, at a temperature of about 50 to 70 ° C,

  In order to produce a thick extract (containing about 10 to 30% (w / w) water).

  The duration of the concentration under reduced pressure depends on the applied reduced pressure conditions, but is generally from 30 minutes to 2 hours. In the concentration method under reduced pressure, the temperature must not exceed 90 ° C in order to avoid loss or destruction of the active components contained in the extract. Lyophilization The purified liquid on the column is concentrated under reduced pressure at

  room temperature without heating, then is quickly frozen and stored.

  Freeze-drying can completely eliminate sublimates and residual water, keeping the product at an extremely low temperature, by

  use of a refrigerant at -100 C under vacuum.

  The duration of the drying operation depends on the amount of product to be treated, its area, the temperature of the refrigerant and the capacity of the vacuum pump. The dried substance is generally obtained in one

1 5 period from 12 to 24 hours.

  The present invention also relates to an extract of dried green tea leaves, dried green tea or soluble tea, obtainable by the

  process as defined above.

  The dry powder obtained by the method described above contains epigallocatechin gallate (EGCG), one of the active components of green tea leaves, represented by the following formula:> COH in a proportion of about 50% to 70% by weight relative to the weight of the extract. Since the dry powder obtained by the process described above is not hygroscopic and has a low specific volume, it can be converted into a pharmaceutical composition, especially in the form of powder, granules, fine granules or tablets. , by

usual processes.

  The dry powder obtained by the process described above is not

  hygroscopic, has a low specific volume and low toxicity.

  The subject of the present invention is therefore also an antithrombotic pharmaceutical composition containing, as active ingredient, at least one green tea extract obtainable by the process described

  previously and at least one pharmaceutically acceptable adjuvant.

  According to a particular embodiment of the invention, the composition

  It is intended to be administered orally.

  The composition according to the invention can also be administered by injection. Among the pharmaceutically acceptable adjuvants, there may be mentioned vehicles such as starch, lactose, calcium carbonate and calcium phosphate, binders such as starch, gum arabic, carboxymethylcellulose, hydroxymethylcellulose and crystalline cellulose, lubricants such as magnesium stearate and talc, separating agents such as calcium carboxymethylcellulose, talc and synthetic aluminum silicate, and pharmaceutically acceptable diluents such as

  than water and vegetable oils.

  The pharmaceutical composition as defined above can be

  presented in different forms by usual methods.

  Among these, there may be mentioned powders, granules, microgranules, tablets, capsules, soft capsules and

Liquids.

  The following examples are not intended to limit this

  invention, but to illustrate it in detail.

  We will now give, by way of illustration, examples of

  preparation of green tea extract and pharmaceutical compositions.

  EXAMPLES OF EXTRACT PREPARATION OF THE GREEN

Example 1

  Extract 200 g of dried green tea leaves, dried green tea or soluble green tea at a temperature of 70 C to 90 C with 4000 ml of hot water for 20 minutes. Filter the extract obtained on filter paper, in order to separate the insoluble substances, then centrifuge for 10 minutes at

2000 rpm.

  Collect the filtrate (approximately 3500 ml) and concentrate under reduced pressure

   C in a rotary evaporator under vacuum to obtain 400 ml of extract.

  Dissolve 400 ml of extract in 400 ml of chloroform to complete

  The elimination of non-polar compounds. Repeat the elimination step

  extraction above more than three times. Extract the aqueous phase three times with 400 ml of ethyl acetate to obtain an organic phase of about 1200 ml and completely concentrate the substance obtained at 50 C under vacuum, in a rotary evaporator, to 50 ml. Add 200 ml of acetone or dioxane to the concentrate and reconcentrate to 20 ml. Inject 20 ml of the concentrate at the top of the Sephadex LH-20 column (inside diameter 35 mm, length 550 mm,

  net weight of Sephadex 200 g). First, rinse with 3000 ml of water-

  acetone or water-dioxane (85:15, v / v) until complete elution of the yellow-green or brown color phase, and then begin to elute with a water-acetone or water-dioxane solution (40:60, v / v). When the yellow-brown or yellow band, which was absorbed at the top of the column, starts to elute, collect the fraction and concentrate it to 50 ml at 70 ° C. by evaporation under vacuum. Store the concentrate overnight at a temperature below -20 C and freeze-dry to obtain 8.6 g of dry powder (proportion

Residual EGCG: 70%).

  The residual proportion of EGCG in the dry powder can be calculated

by the following equation.

  Proportion of EGCG (%) = peak area comprising EGCG / total area of peaks x 100 Dissolve 5.0 mg of dry powder obtained by the above process in 5.0 mg of methanol, and inject 5 JI of the solution obtained in a high-speed liquid chromatography apparatus to determine

the content of EGCG.

  The conditions for the determination by high-speed liquid chromatography are as follows:

  Apparatus: high-speed liquid chromatography apparatus (P-

  6330, Hitachi, Japan) Eluent: mixed solvent acetonitrile-ethyl acetate-phosphoric acid 0.05% (12: 2: 86, v / v / v) Column: Cosmosil 5C18 (inner diameter 4.6 mm, length 150 mm) (Nakalai Tesque Co., Japan) Column temperature: 40 C

Wavelength: 280 nm.

  Under the conditions above, the residence time of the EGCG is

6.9 minutes.

  As indicated by the results of the above measures, the proportion of

remaining EGCG is 70%.

  The proportions of EGCG in the dry powder obtained in the

  Examples 2-6 are determined by the same method as above.

Example 2

  Extract 1.5 kg of dried green tea leaves, dried green tea or soluble green tea with 20 liters of water-ethanol solution (90:10, v / v) at 70 ° C for 20 minutes. Filter the extract obtained on a filter paper and separate the insoluble substances. Collect the filtrate (about 19.0 liters) and concentrate under reduced pressure at 70 ° C. in a rotary evaporator under vacuum to obtain a thick extract. Dissolve the viscous extract in 2.0 liters of hot water, divide it into 500 ml aliquots and extract with 500 ml of chloroform-n-hexane (80:20, v / v) to completely remove the compounds.

  non-polar. Repeat more than three times the removal-extraction step above.

  Extract the aqueous layer three times with 500 ml of ethyl acetate to obtain an organic phase of about 1500 ml and evaporate to dryness in a rotary evaporator under vacuum. Add 500 ml of acetone to the dry substance and concentrate to 50 ml. Inject 50 ml concentrate at the top of the Sephadex LH-20 column (inside diameter 70 mm, length 500 mm, net weight Sephadex 400 g). After removal of the soluble substances with 4.0 liters of water, rinse with 3.0 liters of water / acetone solution (70: 30, v / v) until complete elution of the yellow-green colored phase or brown, and then begin to elute with a water-acetone solution (40: 60, v / v). When the yellow-brown or yellow band, which was absorbed by the top layer of the column, begins to elute, collect the fraction and concentrate up to 100 ml at 70 ° C in a vacuum evaporator. Keep the concentrate overnight at a temperature below -20 ° C and then dry by lyophilization to

  obtain 43.8 g of dry powder (residual proportion of EGCG: 54%).

Example 3

  Extract 200 g of dried green tea leaves, dried green tea or soluble green tea with 4 liters of hot water at a temperature of 70 ° C to 90 ° C for 20 minutes. Filter the extract obtained on a filter paper and separate the insoluble substances, then centrifuge the filtrate at 2000 rpm for minutes to remove the precipitate residue. Collect the higher filtrate (approximately 3500 ml) and concentrate under reduced pressure at 70 ° C in a rotary evaporator under vacuum to obtain 400 ml of extract. Extract the aqueous phase three times with 400 ml of ethyl acetate to obtain an organic phase of about 1200 ml and dry evaporate the organic phase at 50 C to 50 ml in a rotary evaporator under vacuum. Add to the dry substance 200 ml of isopropanol and concentrate to 20 ml. Inject 20 ml of concentrate at the top of the Sephadex LH-20 column (inside diameter mm, length 550 mm, net weight of Sephadex 200 g). Rinse with 3.0 liters of water / acetone solution (85:15, v / v) until complete elution of the yellow-green or brown color phase, and then begin to elute with 0 liters of water solution acetone (55:45, v / v). When the yellow-brown or yellow band, which was absorbed by the top layer of the column, starts to elute, collect the fraction and concentrate it to 50 ml at 70 ° C in a vacuum evaporator. The Sephadex LH-20 column can be reused by eluting residual nonpolar substances on the column with 2.0 liters of acetone. Keep the concentrate overnight at a temperature below -20 ° C. and dry by lyophilization to obtain 14.7 g of dry powder.

  (residual proportion of EGCG: 62%).

Example 4

  Extract 100 g of dried green tea leaves, dried green tea or soluble green tea with 1.0 liter of hot water at a temperature of 70 ° C to 90 ° C for 10 minutes. Filter the extract obtained on a filter paper and separate the insoluble substances. Eliminate the precipitate by centrifugation at 2000 rpm for 10 minutes. Collect the upper filtrate (about 800 ml) and concentrate under reduced pressure at 70 ° C in a rotary evaporator under vacuum to obtain 100 ml of extract. Extract the aqueous phase three times with 100 ml of ethyl acetate to obtain an organic phase of approximately 280 ml and evaporate to dryness to dryness.

   C until a viscous state is reached in a rotary evaporator under vacuum.

  Add to the dry substance 150 ml of methanol and concentrate to 15 ml.

  Inject 15 ml of concentrate at the top of the Sephadex LH-20 column

  (inner diameter 35 mm, length 550 mm, net weight of Sephadex 200 g).

  Rinse with 4.0 liters of water / acetone solution (85:15, v / v) until complete elution of the yellow-green or brown color phase, and then start

  elute with water-acetone solution (30:70, v / v). When the yellow band

  Brown or yellow, which was absorbed by the top layer of the column, begins to elute, collect the fraction and concentrate up to 30 ml at 70 ° C in a vacuum evaporator. Keep the concentrate overnight at a temperature below -20 ° C and then dry by lyophilization to obtain 5 g of dry powder (residual proportion of EGCG: 58%).

Example 5

  Extract 200 g of dried green tea leaves, dried green tea or soluble green tea with 4.0 liters of hot water at a temperature of 70 ° C to 90 ° C for 20 minutes. Filter the extract obtained on a filter paper and separate the insoluble substances. Eliminate the precipitate by centrifugation at 2000 rpm for 10 minutes. Collect the higher filtrate (approximately 3500 ml) and concentrate under reduced pressure at 70 ° C in a rotary evaporator under vacuum to obtain 400 ml of extract. Extract the aqueous phase three times with 400 ml of ethyl acetate to obtain an organic phase of about 1200 ml and evaporate to dryness at 50 ° C. until 50 ml of concentrate are obtained in a rotary evaporator under vacuum. Add 200 ml of methanol to the dry substance and concentrate to 20 ml. Inject 20 ml concentrate at the top of the Sephadex LH-20 column (inside diameter 35 mm, length 550 mm, net weight Sephadex 200 g). Rinse with 2.5 liters of water / isopropanol solution (90:10, v / v) until complete elution of the yellow-green colored phase or

  brown, and then begin to elute with 5 liters of a water-

  isopropanol (50:50, v / v). When the yellow-brown or yellow band, which was absorbed by the upper layer of the column, begins to elute, collect

  fraction and concentrate to 40 ml at 70 ° C in a vacuum evaporator.

  Keep the concentrate overnight at a temperature below 20 C and then dry by freeze-drying to obtain 10.2 g of dry powder

(EGCG proportion: 70%).

Example 6

  Extract 200 g of dried green tea leaves, dried green tea or soluble green tea with 4.0 liters of water-methanol (50:50, v / v) at a temperature of 65 ° C for 60 minutes. Filter the extract obtained on a filter paper and separate the insoluble substances. Eliminate the precipitate by centrifugation at 2000 rpm for 10 minutes. Collect the higher filtrate (about 3000 ml) and concentrate under reduced pressure at 70 ° C in a rotary evaporator under vacuum to obtain a completely dry extract. Dissolve the dry substance in 500 ml of water-methanol (80:20, v / v), extract three times with 400 ml of ethyl acetate to obtain an organic phase of approximately 1200 ml and then concentrate to 50 ml at 50 ° C in a rotary evaporator under vacuum. Add 200 ml of dioxane to the concentrate and concentrate again to 20 ml. Inject ml of concentrate at the top of the Sephadex LH-20 column (inside diameter 35 mm, length 550 mm, net weight Sephadex 200 g). Rinse with 3.0 liters of water / dioxane solution (85:15, v / v) until complete elution of the yellow-green or brown color phase, and then begin to elute with a water-dioxane solution (40: 60, v / v). When the yellow-brown or yellow band, which was absorbed by the upper layer of the column, starts to elute, collect the fraction and then concentrate it to 50 ml at 70 ° C. in a vacuum evaporator. Keep the concentrate overnight at a temperature below -20 C and then dry by lyophilization to obtain

  7.8 g of dry powder (residual proportion of EGCG: 55%).

  The platelet anti-agglutination activity of the fraction obtained above is measured. Blood coagulation is roughly divided into a first hemostasis by platelet action (or platelet time of hemostasis), the platelets being active components of the blood, and in a second hemostasis

  (or plasma time), involving plasma with the exception of platelets.

  As a basic treatment, it is interesting to develop the first hemostasis inhibitory agent to prevent thrombogenesis and blood clotting. In the present invention, dried tea refers to a semi-finished product which is obtained prior to the preparation of the final green tea by thin slicing.

green tea leaves.

PHARMACOLOGICAL STUDY

  In this study, reference is made to the accompanying drawings, in which: FIGS. 1 to 4 are graphical representations, comparing the effect of the antithrombotic agent according to the present invention on agglutination induced by different agglutination promoters, ADP, epinephrine, collagen and ristocetin respectively, FIG. 5 is a graphical representation of the antithrombotic effect of the antithrombotic agent according to the present invention on agglutination, when administered. together with epinephrine and collagen, FIG. 6 is a graphical representation of bleeding time when administering the antithrombotic agent according to the present invention or aspirin as a control, FIG. 7 is a graphical representation of the time of coagulation of whole blood when administering the antithrombotic agent according to the present invention or aspirin as a control, and FIG. plasma clotting time when the antithrombotic agent is administered according to the present invention.

  invention or aspirin as a control.

  Effects in the first stage of hemostasis 1. Platelet Agglutination Inhibition Assay The platelet anti-agglutination activity is measured by the Born turbidimetric method, using an Aggrecorder II PA-3220 apparatus. (Kyoto Daiichi Kagaku Co., Ltd.). The principle of the method is that platelet-rich plasma (PRP) in which the platelet ratio is adjusted to 200,000-400,000 / mm 3 is prepared, agglutination agents or promoters are added. agglutination (ADP, epinephrine, collagen, ristocetin) and the variation in optical density due to agglutination is recorded. Blood is collected from a healthy person with normal platelet characteristics and inserted into a tube containing

  3.8% sodium citrate as anticoagulant, in a ratio of 1: 9.

  A centrifugation of 10 minutes is carried out at 160 g (1000 rpm) and

  obtains PRP in which the proportion of platelets is 200 000-

  400,000 / mm3. The centrifugation is repeated for 10 minutes at 2000 g (3400 rpm) and platelet poor plasma (PPP) is obtained. An Aggrecorder II PA-3220 is used to perform the measurements (37 C, 1000 rpm, correction with platelet-poor plasma). After transfer to the meter, the platelet-rich plasma and the test sample are allowed to stand for 3-5 minutes, then platelet aggregation enhancing agents are added, and the procedure is evaluated. agglutination for minutes. As agglutinating agent (or agglutination promoter), use is made of ADP, epinephrine, collagen and ristocetin. As shown in the above test, the green tea extract has a potent inhibitory effect on platelet agglutination caused by the agglutination promoters at a concentration of 1 mg / ml. The inhibitory effect is more particularly 60% when the agglutination promoter is ADP, 96.5% when the agglutination promoter is epinephrine, 35.5% when the agglutination promoter is the collagen and 36.5% when the agglutination promoter is ristocetin. In contrast, aspirin (0.1 mM) used as a control showed 13.5% inhibition for ADP, 79.5%

  for epinephrine, 72.6% for collagen and zero for ristocetin.

  FIG. 1 is a graphic representation of the antagglutination effect of an antithrombotic agent according to the invention when ADP is injected as

agglutination promoting agent.

  FIG. 2 is a graphic representation of the antagglutination effect of an antithrombotic agent according to the invention when epinephrine is injected.

  as an agent promoting agglutination.

  FIG. 3 is a graphical representation of the antagglutination effect of an antithrombotic agent according to the invention when collagen is injected

  as an agent promoting agglutination.

  FIG. 4 is a graphic representation of the antagglutination effect of an antithrombotic agent according to the invention when ristocetin is injected.

  as an agent promoting agglutination.

  The results of the test indicate that the antithrombotic agent

  The invention has a potent platelet anti-agglutination action.

  2. Determination of antithrombotic activity The induction of an experimental thrombus is carried out according to the method of Dimino et al. For the experiment, ICR male rats having a body weight of 20-30 g are used. Thrombus is induced by injection of a platelet agglutination promoter (epinephrine, collagen) into the caudal vein of the rat, rapidly causing thrombus formation in a capillary vessel. The agglutination accelerating substance is prepared in this test by mixing 15 μl of collagen, 400 mM of epinephrine and 100 μl of physiological saline. The substance is injected into the caudal vein of rats at 100 Rl per 20 g of body weight. Two hours before the start of the test, rats were injected with 100 mg and 10 mg green tea extract fractions per kg of body weight to study the antithrombotic action of green tea extracts. The total volume injected should not exceed 0.4 ml. The antithrombotic effect of green tea extracts is calculated as a percentage of surviving rats, having escaped the death or paralysis of the hind paw that may occur during the injection of a platelet agglutination promoter. Paralysis is defined here by the loss of the ability to use the hind paw for 15 minutes or by continuous tremor. In the test conducted to determine the actual volume of thrombotic induction by epinephrine and collagen, the mixture of 15 111 collagen, 400 mM epinephrine and 100 II physiological saline is able to induce a thrombus of 95%. Thrombus production or its absence is determined by the death or paralysis of the hind paw for 15 minutes after injection of the platelet agglutination promoter. Paralysis of the hind paw is measured by the two symptoms of crawling and trembling. When 100 mg / kg of green tea extract is injected into the abdominal cavity, a preventive effect of 66% is observed, and at 10 mg / kg a preventive effect of 44%. In addition, aspirin

  (100 mg / kg) used as a control has a preventive effect of 45%.

  FIG. 5 is a graphic representation of the antithrombotic effect when epinephrine and collagen are administered as promoters

  agglutination, and then an antithrombotic agent according to the invention.

  According to this test, the antithrombotic agent according to the present invention has a strong antithrombotic effect which is identical to that of aspirin, but with an amount which is only one-tenth of the amount of aspirin. 3. Blood Coagulation Assay (1) Determination of bleeding time Sprague-Dawley rats with a body weight of 180-200 g were administered an experimental substance once a day for 10 days. The rats are anesthetized by intraperitoneal injection of a solution of sodium pentobarbital (400 mg / kg). The tail of the anesthetized rat is cut to a length of 5 mm from the end and the cut tail immersed in 5 cm 3 of saline solution at 37.5 C. The time elapsed between excision of the

tail and hemostasis.

  The results of the test show that the bleeding time increases with the volume administered: 113.2 + 19.6 sec for 10 mg / kg, 250.7 + 17.3 sec per 100 mg / kg, 509 , 8 +1.6 sec for 300 mg / kg in the

  experimental group, versus 65.0 12.9 sec in the control group.

  In the case of administration of aspirin (100 mg / kg), this time is greater than that of the control group and is 165.1 ± 34.3 sec. The results are shown in Figure 6. This is a graphical representation of the bleeding time when administering the antithrombotic agent according to the

  present invention and aspirin as a control.

  (2) Determination of total blood coagulation time The day following the determination of the bleeding time, the rats are anesthetized with ether and 4.5 ml of cardiac blood is removed using a plastic syringe. containing 0.5 ml of a solution of 3.13% sodium citrate. The carefully collected blood is mixed into the syringe, 1 ml of blood is placed in a glass test tube and 200 μl of 1.7% CaCl 2 -2H 2 O are added thereto, and the time required for stirring is measured.

  the formation of coagglutination at room temperature.

  The results of the test show that the coagulation time of whole blood increases with the volume administered: 130.1 +20.5 sec for mg / kg, 140.9 +14.2 sec for 100 mg / kg, 150 , 6 +17.6 sec for 300 mg / kg in the experimental group, against 109.4 + 14.0 sec in the control group. In addition, the administration of aspirin (100 mg / kg) as a control resulted in a time of 137.2-19.4 sec. The results are shown in Figure 7, which is a graphical representation of the total blood clotting time at

  administering an agent according to the invention and aspirin.

  (3) Determination of Plasma Coagulation Time Plasma is obtained from the blood collected in the above test by centrifugation at 25,000 rpm. A 100 μl sample of plasma is mixed with 50 μl of 50 mM CaCl 2 in a test tube and stirred at 37 ° C. The time elapsing between the addition of CaCl 2 and the appearance of the clot is determined. The results of the test show that the plasma coagulation time increases with the volume administered: 177.5 37.6 sec for 10 mg / kg, 219.8 ± 57.9 sec for 100 mg / kg, 233 , 1 + 83.2 sec for 300 mg / kg in the experimental group, against 146, 2 +26.5 sec in the control group. In addition, administration of aspirin (100 mg / kg) as a control resulted in a time of 222.5 ± 40.2 sec. The results are shown in FIG. 8. This is a graphical representation of the plasma clotting time during

  administering an agent according to the invention and aspirin.

  4. Inhibition of lipid hyperoxidation and enzymatic activity in the plasma a) Determination of the inhibitory effect by the production of malonic dialdehyde (MDA) 1 ml of a suspension of platelets is introduced into an isotonic solution of green tea extract (final concentration 1 mg / ml) and added Ca ++ and collagen. Then 2 ml of TBA are added to the resulting mixture and then heated for 20 minutes at 90 ° C., cooled to room temperature and centrifuged for 10 minutes at 3000 rpm. The optical density at 535 nm of the red solution at the top of the precipitated platelets is measured, and the amount of MDA produced is calculated based on the molar absorption index of the MDA-TBA conjugate of 1.56. x 105,

  Indicating that green tea extract inhibits lipid hyperoxidation.

  The amount of MDA produced by the reaction of the secondary product of lipid hyperoxidation can be used as a criterion for the determination of total lipid hyperoxidation. Green tea has similar results to those obtained with aspirin, which inhibits the action of phospholipase A2 inhibitors such as primaquine, quinacrine and oxygenase. d) Determination of the arachidonic acid release activity Green tea extract (final density 1 mg / ml), Ca ++ and collagen are introduced into 1 ml of a platelet suspension and incubated at 37.degree. C. After 30 minutes, the platelets are rapidly removed by filtration through a filter membrane and the filtrate is acidified to a pH below 3. The solution obtained is extracted twice with 2 ml of extraction solvent (chloroform: methyl alcohol). = 1: 1, v / v). The chloroformic bottom layer obtained by centrifugation is transferred to the other test tube and dried with nitrogen gas. The dry substance is dissolved in 50 μl of dimethylformamide (DMF) and 50 μl of DMF solution containing 12 mM monodansylcadaverine and 2 Nul diethylphosphorocyanidate are added to the resulting solution and the resulting mixture is left at room temperature for 15 minutes. minutes, so that arachidonic acid is labeled

by fluorescence.

  The fluorescently labeled arachidonic acid was eluted with a linear gradient of 50% methanol: acetonitrile on the inverted-image column, and the detection was carried out at a wavelength of 340 nm and at a length of

520 nm fluorescence wave.

  As indicated by the quantitative displacement of arachidonic acid, the modification of arachidonic acid caused by green tea extract is less than that caused by primaquine and quinocrine. Accordingly, it is believed that green tea extract avoids lipid hyperoxidation and platelet agglutination by inhibition of cyclooxygenase or lipoxygenase rather than phospholipase as an active part of platelets. 6. Therapeutic effect on hyperlipidemia - Antiperoxidation action (in vitro) Green tea extract is added to a liver microsome fraction (1.5 mg protein / ml) of rat, and cysteine (final concentration: 500 gM) and ferrous sulphate (final concentration: 5 gM) and the resulting mixture is incubated for 30 minutes at 37 C. 3 ml of acid are added thereto.

  10% trichloroacetic acid and 2 ml of supernatant are collected after centrifugation.

  A solution of TBA at 0.67% is added thereto, and the solution obtained is activated in boiling water for 15 minutes, and after cooling, the

Optical density.

  7. Study of action on physiological functions and antihypertensive action (in vitro) Inhibition of angiotensin converting enzyme (ACE) ACE is an enzyme acting on renin-angiotensin It plays a decisive role in vivo in the regulation of blood pressure, body fluids and their electrolytic composition. This enzyme converts angiotensin I (ANG I) into active angiotensin II (ANG II) by releasing the dipeptide (hisleu) at the C-terminus of ANG I produced from renin. The enzyme also plays an important role in regulating the circulatory system of living organisms because it activates ANG II in the

  hypertensive system, and inactivates bradykinin in the hypotensive system.

  As a result, it is possible to prove the activity of green tea extract

  as a depressant agent of blood pressure.

  Aspirin is a potent anticoagulant, however, its ingestion

  prolonged is impossible because of the stomach pains it causes.

  On the contrary, the antithrombotic agent according to the invention is found to be devoid of side effects such as gastric embarrassment and produces the same activity.

  as aspirin, and even more.

  Experimental results prove that green tea extract according to

  The invention has a remarkable antithrombotic action.

  Accordingly, the green tea extract according to the invention can be used as antithrombotic agent, thrombogenesis inhibiting agent or agent.

  inhibitor of the cause causing thrombogenesis.

  Acute toxicity test A physiological solution containing green tea extract is administered to ICR rats by the oral and intravenous routes. The LD50 is calculated by the mortality after 72 hours. We apply the calculation method "up and down" (amount and

  descending). The results are as follows.

  Substance Mode Number LD50 Administered Animal Administration mg / kg Intravenous Green Tea Extract 10 10 N Oral 1000 As indicated by the above results, the antithrombotic agent according to

  the present invention has low acute toxicity.

  The green tea extract according to the present invention may be administered at 1.00-2000.00 mg / kg in one to several doses per day, depending on the age, sex and condition of the patient, or according to the precautions to be observed. The green tea extract according to the present invention can be converted into pharmaceutical compositions by addition of usual adjuvants such as vehicles, binders, lubricants, separating agents, diluents, etc.

  EXAMPLES OF PHARMACEUTICAL COMPOSITIONS

  The following examples are examples of composition

  pharmaceutical composition according to the present invention.

  Example 1 tablets green tea extract 10 mg lactose 20 mg starch 20 mg magnesium stearate appropriate amount The above components are converted into tablets containing

  mg per tablet, by a conventional method of preparation.

  Example 2: Capsules (capsules) green tea extract 100 mg lactose 20 mg starch 19 mg talc 1 mg magnesium stearate appropriate amount The above components are divided into 50 mg capsules, by

  a known method of preparation.

  Example 3: liquid composition extracted from green tea 100 mg saccharide isomerized 10 mg honey 500 mg amide of nicotinic acid (pharmacopoeia) 20 mg caffeine anhydride (pharmacopoeia) 30 mg sodium benzoate 70 mg The above components are mixed to give a drug in liquid form, which is packaged in flasks of smoked glass

  ml that is hermetically sealed and then pasteurized.

  Example 4: solution for injection green tea extract 5 mg sterilized water appropriate amount The above components are converted into solution for injection according to a known method of preparation. Then the solution is conditioned in

  2 ml ampoules which are then sealed and sterilized.

  Example 5: soft capsules 1) components introduced into the capsule extracted from green tea 10 mg polyethylene glycol 230 mg glycerin 20 mg 2) components of the dry shell (% by weight) gelatin 52% glycerin 32% Anidrisorb 35/70 12% water 5% The above components are converted into soft capsules by a

classic method of preparation.

  Example 6: granules green tea extract 10 mg lactose 25 mg The above components are converted into granules by means of a

extrusion granulator.

  On the other hand, the green tea extract according to the present invention can be used in the form of compositions in which the green tea extract is mixed with other known pharmaceutical products having a different action. Compositions of this type are within the scope of the invention.

Claims (5)

claims   A process for preparing a green tea extract comprising the steps of: hydrothermally extracting dried green tea leaves, dried green tea or soluble tea with water or a solution of water and a low molecular weight alcohol to remove insoluble substances, concentrate the resulting extract, redissolve the resulting concentrate with water, extrude the resulting solution with an organic solvent to remove the substances non-polar, concentrate the aqueous layer obtained, and reconcentrate the liquid separated from the concentrate by a method of column separation.   2. Extract of dried green tea leaves, dried green tea or soluble tea, obtainable by the process as defined in accordance with claim 1.   1 5 A pharmaceutical composition having antithrombotic action, comprising, as an active ingredient, at least one green tea extract obtainable by the process as defined in claim 1, and at least one   pharmaceutically acceptable adjuvant.   4. Pharmaceutical composition according to claim 3, characterized in that said composition is in the form of a powder, a tablet, a capsule, a liquid, a soft capsule, a   granule or solution for injection.   5. Use of an extract of dried green tea leaves, dried green tea or soluble tea for the manufacture of a pharmaceutical composition   having anti-thrombotic activity.