KR100276979B1 - Composition for platelet aggregation inhibitor containing hesperidin or hesperetin - Google Patents

Abstract

The present invention relates to a composition for inhibiting platelet aggregation comprising hesperidin or hesperetin, and the composition of the present invention includes a pharmaceutical and food composition.

Description

Composition for inhibiting platelet aggregation containing hesperidin or hesperetin {COMPOSITION FOR PLATELET AGGREGATION INHIBITOR CONTAINING HESPERIDIN OR HESPERETIN}

The present invention relates to a composition for inhibiting platelet aggregation comprising hesperidin (C ₂₈ H ₃₄ O ₁₅ , molecular weight 610.55) or hesperetine (C ₁₆ H ₁₄ O ₆ , molecular weight 302.27), and includes a pharmaceutical and food composition. .

Under normal conditions, platelets do not adhere well to the endothelium. However, for a variety of reasons, endothelial cells in the blood vessel wall are injured and the collagen underneath them exposes platelets to be activated by collagen, which releases Ca ++. Releases thromboxane A ₂ and induces more platelet aggregation and thrombus formation (Fuster V. and Verstrate M., 'Hemostasis, thrombosis, fibrinolysis, and cardiovascular Disease' in Heart Disease , Vol. 2 , E. Braunwald (ed), pp 1809-1842, 1997, WB Saunders Co., Phildelphia).

On the other hand, the formed blood clot further promotes platelet aggregation, and indirectly promotes arteriosclerosis by adhering to arterial walls to promote early stage symptoms of arteriosclerosis formed by attachment of smooth muscle cells and macrophages, and formation of blood clots. Blood vessels are blocked by the back, causing heart disease.

Platelets play an important role in the formation of hemostatic plugs in small arteries or veins. Therefore, the interaction between platelets and blood vessels is on the center of thrombus formation, and many drugs have been introduced for the purpose of suppressing adherent platelets. Thromboxane A2 inhibitors, PGG / H synthase inhibitors, thromboxane synthase inhibitors, thromboxane A2 receptor antagonists, for the prevention of cerebral stroke, coronary artery thromboembolism and atherosclerosis, Ticlopidine (Ticlopidine), GP Hb-IHa inhibitors, drugs such as serotonin antagonists have been introduced, but the situation has a problem due to the side effects and increased hemorrhagic tendency of the drug on other organs.

In the case of aspirin, which is most commonly used to prevent blood clots, there is much debate about whether to actually reduce mortality in cardiovascular or cerebrovascular diseases. Therefore, the development of a material that can prevent blood clots without showing a hemorrhagic tendency is required.

If flavonoids specifically inhibit platelet aggregation caused by collagen, it is expected that they can prevent blood clots by specifically interacting with platelets and the vascular wall without causing hemorrhagic tendencies that may interfere with daily life. Can be. If such materials are developed, it may be possible to prevent atherosclerosis in early stages of atherosclerosis, prevent blood clots of coronary artery disease, and prevent cerebral infarction by blood clots.

In patients with atherosclerosis, the main lesions of atherosclerosis are atherosclerosis, which is manifested by increased plasma lipids and hemodynamics. Atherosclerosis is a large mass of lipids, platelets, and fibrous tissues that accumulate in the vascular lining. Atherosclerosis formed in large blood vessels can clog blood vessels, reduce blood flow, and cause some of the atherosclerosis to fall off to close blood vessels in major organs. In brief, the formation process shows that when endothelial cell damage of inflexible blood vessels occurs, platelets and low-density lipoprotein (LDL) accumulate there, and growth factors are secreted from macrophages that have gathered there, so that smooth muscle cells move from the original position into the vessel. Move and multiply.

In patients with atherosclerosis, a combination of drugs that lower blood lipids and drugs that inhibit platelet aggregation may effectively prevent the formation of atherosclerosis. In particular, it is more effective if it can prevent the aggregation of platelets due to collagen exposed during vascular endothelial cell damage, because it can prevent the onset of early atherosclerosis (Steinberg, D., 'Lipoproteins and the pathogenesis of atherosclerosis'). , Circulation, 76 , 508 (1997))

Therefore, many researchers have tried to prevent thrombosis and atherosclerosis by looking for substances that selectively inhibit platelet aggregation. Aspirin inhibits thromboxane A2 by inhibiting cyclooxygenase. Platelets aggregate by many factors, including ADP, collagen, and thrombin. Aspirin seems to partially inhibit their activity. However, aspirin is weak as a thromboprotectant. Ticlopidin or its chemical analogue clopidogrel has been developed as a new drug because it has no inhibitory effect on platelet aggregation in vitro but has a strong inhibitory activity in vivo . These substances are known to selectively inhibit ADP-induced platelet aggregation, and are known to be effective against transient ischemic cerebral stroke or peripheral arterial or ischemic heart disease (Hass, WK et al ., N. Engl. J. Med., 21 , 501 (1989); and Easton, JD, 'Antiplatelet therapy in the prevention of stroke', Drugs, 42 , 39 (1991). Ticlopidine, however, has been shown to suppress bone marrow (McTavish, D. et al. , 'Ticlopidine: An updated review of its pharmacology and therapeutic use in platelet-dependent disorders', Drugs, 40 , 238 (1990)), and increase the amount of total cholesterol ( Hass, WK et al. , Supra) have reported side effects. In clinical trials, 50% of patients reported side effects such as diarrhea, rash, and neutropenia (Dunn CDR, Scrip. Reports, Stroke: Trends, Treatments and Markets , PJB Publications Ltd. pp 133-139 (1995). )).

The present inventors have found that platelet aggregation inhibitors are known to have low selectivity and many side effects, and thus useful for selectively inhibiting platelet aggregation by collagen exposed when the endothelial layer of the vascular endothelial cell wall is damaged. It came to searching for matter. More specifically, hesperidin and hesperetin, which have been isolated from tangerines, have been studied to develop blood flow improving agents and atherosclerosis prevention agents that inhibit fat streak and thrombus formation in the blood vessel wall and have fewer side effects. The present invention has been found to selectively inhibit platelet aggregation induced by the present invention.

An object of the present invention is to provide a pharmaceutical and food composition for inhibiting platelet aggregation, including hesperidin or hesperetin.

In the present invention, there is provided a pharmaceutical composition for inhibiting platelet aggregation, which contains hesperidin or hesperetin as an active ingredient together with a pharmaceutically acceptable carrier.

In addition, the present invention provides a food composition and a beverage composition for inhibiting platelet aggregation, including hesperidin or hesperetin.

The compositions of the present invention may help to improve blood flow by inhibiting platelet aggregation and at the same time inhibiting thrombus formation.

Hesperidin or hesperetine can be extracted from citrus fruits or prepared by known synthetic methods. Citrus fruits in the present invention refers to a mandarin orange, sugar, citrus, orange, lemon, grapefruit or citron. Hesperidin is especially found in grapefruit, sugar and citron. Many of these extraction methods are disclosed in literatures and patents such as the Merck Index.

Hesperidin or hesperetin inhibits platelet aggregation caused by collagen, and thus is effective in preventing and treating arteriosclerosis due to platelet aggregation, as well as improving blood flow. Toxicity studies studied to date have shown that hesperidin or hesperetin is almost nontoxic when administered orally to rats at a dose of 1000 mg / kg, and does not show any side effects on the function of organs including liver.

As a platelet aggregation inhibitor, hesperidin or hesperetin may be mixed with a pharmaceutically acceptable carrier as an active ingredient to prepare a pharmaceutical composition. The pharmaceutical composition may further include conventionally used excipients, disintegrants, sweeteners, lubricants, flavoring agents and the like, and by conventional methods tablets, capsules, powders, granules, suspensions, emulsifiers, syrups, It may be formulated in unit dosage forms or in multiple dosage forms, such as liquid or parenteral preparations.

The composition for inhibiting platelet aggregation containing hesperidin or hesperetine of the present invention as an active ingredient can be parenterally administered or orally as desired, and hesperidin or hesperetin is 0.5 mg to 100 kg / kg of body weight per day. It can be administered in one to several times so as to be administered in an amount of mg, preferably 2 to 10 mg. The dosage level for a particular patient may vary depending on the patient's weight, age, sex, health condition, diet, time of administration, method of administration, rate of excretion, severity of the disease, and the like.

Hesperidin or hesperetine may be added to a food or beverage for the same purpose. Hesperidin and hesperetin may be added for the development of dietary supplements, for example, various foods, meats, beverages, chocolates, snacks, confections, pizzas, ramen noodles, other noodles, gums, ice creams, alcoholic substances. Beverages, vitamin complexes and dietary supplements.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following Examples are only for illustrating the present invention, but the scope of the present invention is not limited to these.

Example 1 Oral Toxicity Test of Hesperidin

Specific pathogens free of 7-8 weeks of age, 6 females weighing 25-29 g and 6 males weighing 34-38 g, temperature 22 ± 1 ° C, humidity 55 ± 5%, illumination 12L It was bred in the animal room of / 12D. Mice were allowed to acclimate for about a week before being used in the experiment. Feed for experimental animals (Jeil Jedang Co., Ltd., mice and rats) and drinking water were supplied after sterilization and free ingestion.

Hesperidine was prepared at a concentration of 100 mg / ml using 0.5% Tween 80 as a solvent, followed by oral administration of 0.2 ml per 20 g of mouse body weight. Samples were administered orally once and observed for side effects or lethality for 10 days after administration. That is, on the day of dosing, changes in general symptoms and the presence or absence of dead animals were observed at least once in the morning, at least 1 pm, 4 hours, 8 hours, 12 hours, and the next day until the 10th day of administration. In addition, the animals were killed and dissected at 10 days of administration, and the internal organs were visually examined. The change in body weight was measured at the interval of 1 day from the day of administration to observe the weight loss phenomenon of the animal by hesperidin.

The purpose of this study was to provide information on available doses in general pharmacology and drug efficacy tests and to derive basic data on toxicity by identifying the degree of acute toxicity in the oral route of hesperidin. Got it.

For acute oral toxicity, no lethal animals were observed for 10 days at a dose of 1,000 mg / kg of hesperidin. An autopsy of the surviving animals was performed 10 days later, and there were no gross lesions, and normal body weight gain was observed without any weight loss for 10 days from the day after the administration.

In conclusion, hesperidin was not toxic upon oral administration at the above concentrations.

Example 2 Hesperidin and Hesperetin Inhibit Platelet Aggregation Effect

When platelets are separated from a patient and agglutinating substances (eg, ADP, epinephrine, collagen, ristocetin, etc.) are added, platelet aggregation occurs and dozens of platelets are aggregated. Platelet aggrometer (Platelet aggrecorder II, PA-3220, Chronolog, USA) is used to compare the absorbance before and after aggregation to determine what percentage of the total cause aggregation. Before the platelet aggregation material was added during the measurement process, the test material was added in advance to incubate the platelets, and after reacting with the aggregation material, the amount of the aggregation inhibitory effect was measured. The measuring method is O'Brein JR., J. Clin. Pathol., 15 , 452 (1962) and Aok et al. (Aok, H. et al., Anesthesiology, 88 , 362 ( 1998 ). ). ADP 100 μM, epinephrine 200 μM, collagen 2 mg / ml or ristocetin 15 mg / ml was used as the coagulant. All reagents used in the test were Sigma Diagnostics, St. Louis, Mo., USA. Purchased).

9 ml of blood was collected from the people and collected in a container containing 1 ml of 3.8% sodium citrate. The sample was centrifuged at 1,000 rpm for 10 minutes and then the supernatant of plasma was collected to adjust the platelet count to 200,000-400,000 / mm ³ . In three cuvettes, platelet-controlled plasma was added to 450 μl and placed in an aggrecoder warmed to 37 ° C., followed by warming for 3 minutes. In this case, except for the first cuvette used as a control, 5 µl of hesperidin or hesperetine solution dissolved in DMSO (dimethyl sulfoxide) at a concentration of 10 mg / l was added as a test substance. Finally, 500 μl of 0.05 μg test substance (1/2 μM hesperidin and 1/6 μM hesperetin, respectively).

To determine the base line, platelet deficient plasma was placed in a cuvette and the absorbance was measured. In three cuvettes containing platelet-rich plasma, 50 μl of 2 mg / ml collagen was added as a coagulation promoter, and then the coagulation reaction was performed. In addition, the same procedure was repeated using 100 μM ADP, 200 μM epinephrine, and 50 μl of 15 mg / ml ristocetin.

As a result, hesperidin and hesperetin inhibited platelet aggregation caused by collagen. However, it did not affect the aggregation reaction by ADP, epinephrine, ristocetin and the like. Therefore, hesperidin and hesperetin selectively inhibit only platelet aggregation induced by collagen, which can be referred to as a reaction by a new mechanism of action compared to existing drugs. The inhibitory effect of aggregation was also slightly different depending on the blood of the patient. Platelet aggregation inhibitory effect of 0-20% was shown in normal control plasma, and platelet aggregation inhibitory effect was 59-93% in hesperidin-treated plasma, and platelet aggregation inhibitory effect was 15-93% in hesperetin-treated plasma. Was observed.

The above results indicate that the administration of hesperidin or hesperetin has an effect of inhibiting platelet aggregation in the blood.

My example

The pharmaceutical composition was prepared using the following ingredients.

Amount (mg / capsule)

Active Ingredients (Hesperidin or Hesperetine) 200

Vitamin C 100

Dry starch 180

Magnesium Stearate 20

500 mg total

Hard gelatin capsules were prepared to inhibit platelet aggregation by thoroughly mixing the ingredients and filling hard gelatine capsules in a total amount of 500 mg.

A composition containing hesperidin or hesperetin prevents the aggregation of platelets due to collagen exposed during vascular endothelial cell damage, thereby preventing and treating the formation of atherosclerosis or atherosclerosis or thrombosis. It can be used as a composition and a food composition.

Claims (2)

A composition for inhibiting platelet aggregation by collagen, comprising an effective amount of hesperidin or hesperetine as an active ingredient together with a pharmaceutically acceptable carrier. The method of claim 1, Wherein the effective amount of hesperidin or hesperetin is 0.5 mg to 100 mg / kg body weight / day.