KR102287390B1 - Pharmaceutical composition comprising the leaf extracts of polygonium tinctorium as an effective component for prevention or treatment of thrombosis and health functional food comprising the same - Google Patents

Abstract

The present invention relates to an antithrombotic composition containing a hot water extract of indigo leaf (Leaf of Polygonium tinctorium ) as an active ingredient, and more particularly, to prevent thrombosis through strong platelet aggregation inhibition or It relates to a pharmaceutical composition for treatment/improvement and a health functional food. The hot water extract of indigo leaf as an active ingredient of the pharmaceutical composition for the prevention or treatment of thrombosis of the present invention and a health functional food, as demonstrated through the Examples of the present specification, shows strong antithrombotic activity by inhibiting platelet aggregation and At the same time, it has little haemolytic activity on human red blood cells, has excellent thermal stability, and no loss of aggregation inhibitory activity even under acidic conditions of pH 2 It is expected that the active ingredient can be used as an extract, and the active ingredient is processed into various forms such as extracts, powders, pills, tablets, etc. .

Description

PHARMACEUTICAL COMPOSITION COMPRISING THE LEAF EXTRACTS OF POLYGONIUM TINCTORIUM AS AN EFFECTIVE COMPONENT FOR PREVENTION OR TREATMENT OF THROMBOSIS AND HEALTH FUNCTIONAL

The present invention relates to an antithrombotic composition containing a hot water extract of indigo leaf (Leaf of Polygonium tinctorium ) as an active ingredient, and more particularly, to prevent thrombosis through strong platelet aggregation inhibition or It relates to a pharmaceutical composition for treatment/improvement and a health functional food.

As a component of the human body, blood has a variety of important functions, such as transporting oxygen, nutrients, and wastes, buffering, maintaining body temperature, osmotic pressure and ion balance, maintaining water schedule, regulating fluidity, maintaining and controlling blood pressure, and defending the body. there is. Normal blood circulation facilitates blood circulation as the blood coagulation and thrombolytic systems in the body are controlled complementary to each other. , it has been reported that the blood coagulation system is activated to form a fibrin clot centered on platelet agglomerates.

On the other hand, the generation of fibrin thrombus is activated by thrombin involved in fibrin coagulation through a multi-step reaction of numerous blood coagulation factors, and finally produces fibrin monomers from fibrinogen. The fibrin monomers are polymerized by calcium, and platelets and endothelium It binds to cells and forms a fibrin polymer cross-linked by factor XIII, forming a permanent thrombus. In addition, thrombin plays a pivotal role in thrombus formation, such as activating platelets, factor V, and factor VII to promote a blood coagulation reaction. Therefore, the thrombin activity inhibitor can be used as a very useful prophylactic and therapeutic agent for various thrombotic diseases caused by excessive blood clotting. On the other hand, it is known that the activation of prothrombin following sequential activation of factor XII, factor XI, factor XII, factor IX, and X in the endogenous thrombus formation pathway ultimately activates thrombin. It has become a target for development of therapeutic agents for sexual diseases, and in the case of an exogenous thrombus formation pathway, thrombus formation according to the activation of factor II (prothrombin), factor V, factor VII, and factor X is known. Until now, various anticoagulants such as heparin, coumarin, aspirin, urokinase, etc., antiplatelet agents, and thrombolytic agents have been used for the prevention and treatment of thrombotic diseases. Its use is limited due to reactions and the like.

On the other hand, indigo plant is the only natural dye material with a beautiful color, excellent dyeing properties for natural fabrics, resistance to acids and alkalis, good fastness to washing, sweat and friction, and excellent functionality such as insecticidal and antibacterial properties. It is a traditional dyeing material (Song SW et al., 2008. J Korean Soc Dyers Finishers, 20, 18-24). In fact, indigo leaf has been used not only as a salt material, but also as a medicine for antipyretic, detoxification, and small swelling in the folklore (Tokuyama-Nakai S et al., 2018. Appl Biochem Biotechnol, 184, 414-431), and recently, the antioxidant activity of indigo leaf extract (Jang HG et al., 2012, Appl Biochem Biotechnol, 167, 1986-2004), colorectal cancer growth inhibition (Micallef MJ, 2002. Int Immunopharmacol, 2, 565-578), anti-inflammatory (Tokuyama-Nakai S et al., 2018. Appl) Biochem Biotechnol, 184, 414-431; Iwaki K et al., 2011, J Ethnopharmacol, 134, 450-459), anti-atopic (Han NR et al., Phytomedicine, 21, 453-600), anti-allergic (Kunikata T et al., Eur J) Pharmacol, 410, 93-100), inhibition of dermatophytes and Helicobacter pylori (Kataoka M et al., 2001, J Gastroenterol, 36, 5-9; Honda G et al., 1980, Planta Med, 38, 275-276), cholesterol synthesis Inhibitory activity (Kimura H et al., 2015. J Pharm Biomed Anal, 108, 102-112) and anti-HIV activity (Zhong Y et al., 2005, Antiviral Res, 66, 119-128) have been reported. However, until now, the strong antithrombotic activity of the indigo leaf extract by inhibition of platelet aggregation is not known.

Patents related to indigo can be divided into a patent on the manufacture of indigo dye, a patent on a dyeing method using the manufactured indigo dye, and a patent on household goods dyed with indigo. First, with respect to the production of indigo dye, Republic of Korea Patent No. 10-0264675 [Method for producing natural indigo dye] and Registered Patent No. 10-0540642 [method of extracting indigo dye from indigo dye] are disclosed, and dyeing technology In relation to this, [Indigo dyeing and coloring apparatus and dyeing method] in Registered Patent No. 10-1619850, and [Indigo dyeing apparatus and dyeing method using natural fermented indigo dye] are disclosed in Registered Patent No. 10-1357105. In addition, as indigo dyeing products, [Eye health eye patch using fermented indigo dyeing] in Registered Patent No. 10-1233697 and [Natural indigo dyeing diaper] are disclosed in No. 10-2005-0079595. However, until now, an antithrombotic active composition of indigo leaf extract is not known.

Tokuyama-Nakai S, Kimura H, Ishihara T, Jisaka M, Yokota K. Appl Biochem Biotechnol. 2018. 184: 414-431.

An object to be solved in the present invention is to provide an antithrombotic composition comprising extracting, separating and purifying an active substance for inhibiting platelet aggregation from indigo leaf, and using it as an active ingredient.

In order to solve the above problems, the present invention provides a pharmaceutical composition for preventing or treating thrombosis containing a hot water extract of Leaf of Polygonium tinctorium as an active ingredient.

Jjokip (Leaf of Polygonium tinctorium) provides a health functional food for the prevention or improvement of thrombosis containing hot water extract as an active ingredient.

Indigo leaf (Leaf of Polygonium tinctorium ) provides a platelet aggregation inhibitor containing hot water extract as an active ingredient.

The hot water extract of indigo leaf as an active ingredient of the pharmaceutical composition for the prevention or treatment of thrombosis of the present invention and a health functional food, as demonstrated through the Examples of the present specification, exhibits strong antithrombotic activity by inhibiting platelet aggregation and At the same time, it has almost no hemolytic activity on human red blood cells, has excellent thermal stability, and no loss of aggregation inhibitory activity even under acidic conditions of pH 2, so it is used for the prevention and treatment of thrombosis such as ischemic stroke and hemorrhagic stroke through improvement of blood circulation It is expected that the active ingredient can be used as an extract, and the active ingredient is processed into various forms such as extracts, powders, pills, tablets, etc. .

1 shows the dried indigo leaf used in the present invention.
Figure 2 shows the human platelet aggregation inhibitory activity of indigo leaf hot water extract: 1: solvent control (DMSO), 2: aspirin (0.25 mg/ml), 3: aspirin (0.125 mg/ml), 4: hot water extract of indigo leaf ( 0.25 mg/ml), 5: Indigo leaf ethanol extract (0.25 mg/ml).

Hereinafter, the present invention will be described in detail.

In order to test the antithrombotic efficacy of indigo leaf, the inventors of the present invention prepared a hot water extract and an ethanol extract prepared by a certain method, respectively, and evaluated the antithrombotic activity of the extract to recover the hot water extract as an antithrombotic component, To utilize the hot water extract as a pharmaceutical composition and health functional food for preventing or treating/improving thrombosis by confirming that the hot water extract has excellent thermal stability and acid stability while exhibiting little hemolytic activity against human red blood cells. did.

Specifically, the present inventors prepared a hot water extract and an ethanol extract from dried indigo leaves in order to develop a pharmaceutical composition and a health functional food for the prevention, treatment/improvement of thrombosis using indigo leaf, which has been used for a long time as a natural dye, respectively. Thrombin Time, Prothrombin Time, and Coagulation Factor Inhibition (Activated Partial Thromboplastin Time: aPTT) were evaluated for the antithrombotic activity of these extracts. In the extract, good prothrombin inhibition, weak thrombin inhibition, and blood clotting factor inhibitory activity were confirmed, and in the ethanol extract, excellent thrombin inhibition, prothrombin inhibition, and blood clotting factor inhibitory activity were confirmed. The ethanol extract effectively inhibited blood coagulation by exhibiting 1.86-fold prolonged thrombin time, 1.79-fold prolonged prothrombin time, and 1.65-fold prolonged aPTT compared to the no-addition group at a concentration of 5 mg/ml, and this activity was aspirin 1.5 mg/ml It was superior to the anticoagulant activity of the concentration. On the other hand, the hot water extract showed a 1.42-fold prolonged prothrombin time compared to the no-addition group at the concentration of 5 mg/ml. On the other hand, the hot water extract exhibited 58% platelet aggregation at a concentration of 0.25 mg/ml, indicating strong platelet aggregation inhibitory activity. In addition, it was confirmed that the hot water extract did not show any hemolytic activity against human red blood cells up to a concentration of 1 mg/ml, and thus did not induce acute toxicity.

Accordingly, the present invention provides a pharmaceutical composition for the prevention or treatment of thrombosis containing the hot water extract of indigo leaf as an active ingredient.

In addition, the present invention provides a health functional food for preventing or improving thrombosis containing hot water extract of indigo leaf as an active ingredient.

In addition, the present invention provides a platelet aggregation inhibitor comprising a hot water extract of indigo leaf as an active ingredient.

Hereinafter, the method for preparing the hot water extract of indigo leaf of the present invention and the efficacy experiment, etc. will be described in more detail.

The inventors of the present invention in order to achieve the object of the present invention, the steps of drying the harvested indigo leaves when the flower buds start to appear; grinding the dried indigo leaves; preparing an extract from indigo leaf powder; Experiments were performed in the step of evaluating the anticoagulant activity and platelet aggregation inhibitory activity of the extract and the step of evaluating the stability of the hot water extract.

The "indigo leaf extract" included in the composition of the present invention can be obtained by extracting the dried indigo leaf with a solvent, filtering the extract using a filtration network of 0.06 mm or less, and concentrating it under reduced pressure.

The solvent used in the present invention is water (cold water, hot water), alcohol, anhydrous or hydrous lower alcohol having 1 to 4 carbon atoms (methanol, ethanol, alcohol, propanol, butanol, etc.), a mixed solvent of the lower alcohol and water, etc. available, and hot water extraction is most preferred.

The indigo leaf extract of the present invention may be prepared as a powder through a conventional powdering process such as drying under reduced pressure, freeze drying, or spray drying. They are not degraded by various degrading enzymes in plasma, and they maintain activity even at 100°C heat treatment and pH 2 in the human stomach.

The active ingredient of the present invention can be used for prevention or treatment of various diseases related to thrombosis. These diseases include, for example, arterial thrombosis, acute myocardial infarction, chest pain, shortness of breath, loss of consciousness, ischemic stroke, hemorrhagic stroke, headache, dyskinesia, paresthesia, personality changes, blurred vision, epileptic seizures, pulmonary thrombosis , deep vein thrombosis, lower extremity edema, pain and acute peripheral arterial occlusion, and the like. Examples of venous thrombosis include deep vein thrombosis, portal vein thrombosis, acute renal vein occlusion, cerebral vein sinus thrombosis, and central retinal vein occlusion.

In addition, the active ingredient of the present invention is used as a pharmaceutical use of a platelet aggregation inhibitor, and may be commonly referred to as an antiplatelet agent.

The pharmaceutical composition comprising the active ingredient of the present invention can be prepared according to a conventional method for each purpose of use, oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., and injections of sterile injection solutions. It can be formulated and used in various forms, such as oral administration, or administered through various routes including intravenous, intraperitoneal, subcutaneous, rectal, topical administration, and the like.

The pharmaceutical composition may further include a carrier, excipient, or diluent, and examples of suitable carriers, excipients or diluents that may be included include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, Starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, amorphous cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil and the like. In addition, the pharmaceutical composition of the present invention may further include a filler, an anti-agglomeration agent, a lubricant, a wetting agent, a flavoring agent, an emulsifier, a preservative, and the like.

In a preferred embodiment, solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient in the pharmaceutical composition, for example, starch, calcium carbonate, It is formulated by mixing sucrose, lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate, talc and the like may be used.

As a preferred embodiment, the oral liquid formulation may be exemplified by a suspension, an internal solution, an emulsion, a syrup, and the like, and various excipients, for example, a wetting agent, a sweetener, Perfumes, preservatives, and the like may be included.

As a preferred embodiment, sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized agents, suppositories, and the like can be exemplified as formulations for parenteral administration. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Injections may contain conventional additives such as solubilizing agents, isotonic agents, suspending agents, emulsifying agents, stabilizing agents, and preservatives.

The active ingredient of the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is determined by the type, severity, activity of the drug, and the type of disease in the patient; Sensitivity to the drug, time of administration, route of administration and rate of excretion, duration of treatment, factors including concomitant drugs, and other factors well known in the medical field. The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or may be administered in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiple times. In consideration of all of the above factors, it is important to administer an amount that can obtain the maximum effect with a minimum amount without side effects, which can be easily determined by those skilled in the art.

In a preferred embodiment, the effective amount of the active ingredient in the pharmaceutical composition of the present invention may vary depending on the age, sex, and weight of the patient, and generally 1 to 5,000 mg, preferably 100 to 3,000 mg per kg of body weight daily Alternatively, it may be administered every other day or divided into 1 to 3 times a day. However, since it may increase or decrease depending on the route of administration, disease severity, sex, weight, age, etc., the dosage is not intended to limit the scope of the present invention in any way.

The pharmaceutical composition of the present invention may be administered to a subject through various routes. Any mode of administration can be envisaged, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection.

In the present invention, "administration" means providing a predetermined substance to a patient by any suitable method, and the administration route of the pharmaceutical composition of the present invention is oral or parenteral through all general routes as long as it can reach the target tissue. It can be administered orally. In addition, the composition of the present invention may be administered using any device capable of delivering an active ingredient to a target cell.

In the present invention, "subject" is not particularly limited, but includes, for example, humans, monkeys, cattle, horses, sheep, pigs, chickens, turkeys, quails, cats, dogs, mice, rats, rabbits or guinea pigs. and preferably a mammal, more preferably a human.

In addition, the health functional food of the present invention can be used in various ways, such as food and beverage effective for preventing or improving thrombosis. Foods containing the active ingredient of the present invention include, for example, various foods, beverages, gums, tea, vitamin complexes, health supplements, and the like, and may be used in the form of powders, granules, tablets, capsules, or beverages. .

In general, the active ingredient of the present invention may be added in an amount of 0.01 to 15% by weight of the total food weight, and the health beverage composition may be added in a ratio of 0.02 to 10g, preferably 0.3 to 1g, based on 100ml.

In addition to containing the above compound as an essential ingredient in the proportion indicated, the health functional food of the present invention may contain food pharmaceutically acceptable food supplement additives, such as natural carbohydrates and various flavoring agents, as additional ingredients.

Examples of the natural carbohydrate include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and conventional sugars such as polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol.

The flavoring agent includes: thaumatin; A natural flavoring agent such as stevia, such as rebaudioside A or glycyrrhizin, and a synthetic flavoring agent such as saccharin or aspartame may be used. The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the health functional food of the present invention. In addition to the above, the health functional food of the present invention includes various nutrients, vitamins, minerals, flavoring agents such as synthetic and natural flavoring agents, colorants and thickeners, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloids It may contain thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonation agents used in carbonated beverages, and the like. In addition, the health functional food of the present invention may contain natural fruit juice, fruit juice for the production of fruit juice drinks, vegetable drinks, and the like. These components may be used independently or in combination. The ratio of these additives is generally selected in the range of 0.01 to about 20 parts by weight per 100 parts by weight of the active ingredient of the present invention.

Hereinafter, the present invention will be described in more detail through specific examples. The following examples are only one preferred embodiment of the present invention, and the scope of the present invention is not limited to the scope of the following examples.

<Example>

Example 1: Preparation of extract of indigo leaf and analysis of its components

After drying the indigo leaves cultivated and harvested in Andong, Gyeongbuk in October 2017 (FIG. 1), the indigo leaf powder was prepared by grinding it into a 50-100 mesh. When preparing the indigo leaf ethanol extract, 10 times the amount of ethanol is added to the sample, and after extraction is repeated 3 times at room temperature, the extract is collected, filtered through filter paper (Whatsman No. 2), and concentrated under reduced pressure (Eyela Rotary evaporator N-1000, Tokyo Rikakikai) Co., Ltd. Japan) to prepare a powder to prepare an ethanol extract. When preparing a hot water extract, 20 times distilled water was added to the dried indigo leaf, extracted at 100° C. for 1 hour, and filtered and reduced pressure in the same manner as above. It was concentrated to prepare a powder. Each extraction yield and the component analysis results of the extracts are shown in Table 1. The content of total polyphenols, total flavonoids, total sugars and reducing sugars was determined by component analysis. For the total polyphenol content, 50 μl of Folin-ciocalteau and 100 μl of Na ₂ CO ₃ saturated solution were added to 400 μl of the extraction sample, left at room temperature for 1 hour, and absorbance was measured at 725 nm. As a standard reagent, tannic acid was used. For the total flavonoid content, each sample was extracted with methanol for 18 hours, and 4 ml of 90% diethylene glycol was added to 400 μl of the filtered extraction sample solution, 40 μl of 1N NaOH was added again, and the absorbance was measured at 420 nm after reaction at 37° C. for 1 hour. As a standard reagent, rutin was used. Reducing sugar was quantified by DNS method and total sugar was quantified by phenol-sulfuric acid method.

[Table 1] Analysis of yield and components of indigo leaf extract

As shown in Table 1, the extraction efficiency of the hot water extract was 27.6%, which was about 2 times higher than that of the ethanol extraction, and the indigo color was clearer of the ethanol extract. As a result of measuring the total polyphenol content of each extract, the hot water extract showed a significantly higher 51.2 mg/g than the ethanol extract, but specifically, the total sugar content (297.8 mg/g) of the ethanol extract was 1.2 times higher than that of the hot water extract. On the other hand, total flavonoid and reducing sugar contents were similar in hot water and ethanol extracts (p<0.05). These total polyphenols and total flavonoid contents were found to be 2-3 times higher than the previously reported contents of indigo leaves, but this is considered to be the result of being affected by the harvest time, maturity and extraction solvent of the indigo leaf used.

Example 2: Anticoagulant activity of indigo leaf extract

The blood coagulation inhibitory activity of various indigo leaf extracts prepared in Example 1 was evaluated, and previously reported methods (Sohn et al., 2004. Kor. J. Pharmacogn 35. 52-61; Kwon et al., 2004. J. Life Science, 14. 509-513; Ryu et al. 2010. J. Life Science, 20. 922-928), thrombin time, prothrombin time, and AP time were measured and evaluated. For plasma, commercially available control plasma (MD Pacific Technology Co., Ltd, Huayuan Industrial Area, China) was used, and thrombin time, prothrombin time, and AP time were measured as follows.

Thrombin Time

At 37°C, 50 μl of 0.5U thrombin (Sigma Co., USA), 50 μl of 20 mM CaCl ₂ , and 10 μl of sample extracts of various concentrations were mixed in a tube of Amelung coagulometer KC-1A (Japan) and reacted for 2 minutes, and then 100 μl of plasma was collected. After addition, the time until plasma coagulation was measured. Aspirin (Sigma Co., USA) was used as a control, and DMSO was used instead of the sample as a solvent control. DMSO showed a coagulation time of 30.5 seconds. The thrombin inhibitory effect was expressed as the average of three or more repeated experiments, and the thrombin inhibitory activity was expressed as the value obtained by dividing the coagulation time at the time of sample addition by the coagulation time of the solvent control.

prothrombin time

70 μl of standard plasma (MD Pacific Co., China) and 10 μl of sample solutions of various concentrations were added to the tube of Amelung coagulometer KC-1A (Japan), heated at 37° C. for 3 minutes, 130 μl of PT reagent was added, and plasma was coagulated. The time until completion was expressed as the average value of the experiment repeated three times. Aspirin (Sigma Co., USA) was used as a control, and DMSO was used instead of the sample as a solvent control. DMSO showed a coagulation time of 16.7 seconds. The prothrombin inhibitory activity was expressed as a value obtained by dividing the coagulation time at the time of sample addition by the coagulation time of the solvent control.

Activated Partial Thromboplastin Time (aPTT)

100 μl of plasma and 10 μl of sample extracts of various concentrations were added to the tube of Amelung coagulometer KC-1A (Japan) and heated at 37° C. for 3 minutes. Then, 50 μl of aPTT reagent (Sigma, ALEXIN ^TM ) was added, and 3 Incubated for minutes. Then, after _{adding 50 μl CaCl 2} (35 mM), the time until plasma coagulation was measured. As a solvent control, DMSO was used instead of the sample, and in this case, the clotting time was 58.1 seconds. The results of aPTT were expressed as the average value of the experiment repeated three times, and the blood coagulation factor inhibitory activity was expressed as the value obtained by dividing the aPTT at the time of sample addition by the aPTT of the solvent control.

[Table 2] Blood clotting inhibitory activity evaluation of indigo leaf extract

As a result, as shown in Table 2, aspirin used clinically at a concentration of 1.5 mg/ml showed 1.64 times longer thrombin time, 1.31 times longer prothrombin time, and 1.60 times longer aPTT compared to the no-addition group, thereby preventing blood coagulation. It was confirmed that the inhibition was effective. On the other hand, the hot water extract of indigo leaf showed good prothrombin inhibition, weak thrombin inhibition, and blood coagulation factor inhibition, and the prothrombin time was increased by 1.42 times compared to the non-additive group at a concentration of 5 mg/ml. Indigo leaf ethanol extract showed excellent thrombin, prothrombin, and blood coagulation factor inhibitory activity. At 5 mg/ml, the thrombin time, 1.79 times longer prothrombin time, and 1.65 times aPTT were exhibited, compared to the no-addition group, resulting in blood coagulation. was effectively inhibited, and this activity was superior to the anticoagulant activity of 1.5 mg/ml aspirin.

Example 3: Human platelet aggregation inhibitory activity of indigo leaf extract

The human platelet aggregation inhibitory activity was evaluated for the indigo leaf extract prepared in Example 1, and the results are shown in Table 3 and FIG. 2 . Platelets are disk-shaped small cells that circulate in blood vessels together with various blood cells. Instead of having a nucleus, they have cytoplasmic granules containing various substances related to vascular damage protection and platelet aggregation in high concentrations. It is an important cell that secretes factors and initiates thrombus formation by binding to collagen exposed due to endothelial cell damage to form a primary hemostatic plug. Therefore, platelet aggregation inhibition is a very important activity to prevent thrombus formation. The platelet aggregation inhibitory activity was evaluated according to the following method.

Platelet aggregation inhibition activity

Platelets were human concentrated platelets, which were washed once with washing buffer (138 mM NaCl, 2.7 mM KCl, 12 mM NaHCO ₃ , 0.36 _{mM NaH 2} PO ₄ , 5.5 mM Glucose, 1 mM EDTA, pH 6.5). Then, resuspended in suspending buffer (138mM NaCl, 2.7mM KCl, 12mM NaHCO ₃ , 0.36mM NaH ₂ PO ₄ , 5.5mM Glucose, 0.49mM MgCl ₂ , 0.25% gelatin, pH 7.4), and then at 3,000 rpm for 10 minutes After centrifugation, it was resuspended in suspending buffer, and the platelet count was adjusted to be ^{4x10 9 /ml.} Then, 2.5 μl collagen was added to the 1 ml suspension, reacted for 5 minutes, and platelet aggregation was measured at 37° C. using a whole-blood aggregometer (Chrono-log, USA).

[Table 3] Human platelet aggregation inhibitory activity of indigo leaf extract

As a result, as shown in Table 3 and FIG. 2, in the case of DMSO as a solvent control, platelets aggregated quickly and strongly by the addition of collagen, and aspirin, a platelet aggregation inhibitor, strongly inhibited platelet aggregation in a concentration-dependent manner. At this time, aspirin showed an aggregation degree of 19.4% at a concentration of 0.25 mg/ml and a degree of aggregation of 32.6% at a concentration of 0.125 mg/ml, confirming the evidence for its use as an antithrombotic agent in clinical practice. On the other hand, the hot water extract of indigo leaf showed a degree of aggregation of 58.3% at the concentration of 0.25 mg/ml, showing excellent platelet aggregation inhibitory activity, but the ethanol extract of indigo leaf showed 235.7% of platelet aggregation at the concentration of 0.25 mg/ml, rather reducing the formation of blood clots. has been shown to promote Since the effect of inhibiting platelet aggregation and promoting platelet aggregation of indigo leaves is not known so far, it is suggested that hot water extract of indigo leaf can be developed as an antithrombotic agent according to aggregation inhibition, and ethanol extract can be developed as a hemostatic agent by promoting aggregation.

Example 4: Evaluation of Plasma, Acid and Thermal Stability of Indigo Leaf Extract

Plasma stability, thermal stability, and acid stability with respect to platelet aggregation inhibitory activity were confirmed for the hot water extract of indigo leaf obtained in Example 1 above. The active fraction maintained excellent activity without loss of aggregation inhibitory activity even after 1 hour heat treatment at 100° C., 1 hour treatment at pH 2 (0.01 M HCl), and 1 hour treatment in plasma. The above results suggest that the hot water extract prepared from indigo leaf grown in large quantities as a natural salt material can be used as an antithrombotic agent, and it is judged that it can supplement and replace aspirin, which has side effects such as gastrointestinal disturbances.

Claims (3)

delete delete Inhibitor of platelet aggregation containing leaf of Polygonium tinctorium hot water extract as an active ingredient.

Images