KR20100079786A - Antiplatelet agent containing caffeic acid and chlorogenic acid for elevating the production of camp and cgmp - Google Patents

Abstract

PURPOSE: An antithrombotic composition containing caffeic acid or chlorogenic acid is provided to enhance generation amount of cAMP and cGMP. CONSTITUTION: An antithrombotic agent which enhances generation amount of cAMP(cyclic adenosine monophosphate) contains caffeic acid or chlorogenic acid. The caffeic acid and chlorogenic acid are denoted by chemical formula 1 and 2, respectively. The increase of cAMP and cGMP induces reduction of Ca^2+ concentration.

Description

Antiplatelet agent containing caffeic acid and chlorogenic acid for elevating the production of cAMP and cGMP, containing caffeic acid and chlorogenic acid as active ingredients

The present invention relates to a composition having an antiplatelet action, and more particularly, to an antiplatelet agent containing caffeic acid and chlorogenic acid to increase the production of cAMP and cGMP, which are platelet aggregation inhibitors.

Cardiovascular disease is caused by atherosclerosis and thrombosis. Thrombosis is a disease caused by excessive platelet aggregation due to activation of platelets. In recent years, as interest in the inhibition of platelet function has increased, there has been a growing interest in preventing thrombosis. In particular, research on the prevention of cardiovascular disease and stroke of polyphenols has been known for the past 15 years. The prevention of diseases caused by food intake is made from artificial synthetic products, which is much more stable than drugs with many side effects. In recent years, as the importance of plant foods has been highlighted, positive effects on the vascular function and platelet function of foods rich in polyphenol compounds have been reported.

Polyphenols are abundant in plants and important for human and animal food intake, from simple phenolic acids (e.g. hydroxycinnamate) containing one phenolic ring to phenolic rings. It refers to everything from dogs to complex polymers (eg, flavonoids and tannins). Polyphenols are very important for plants and are involved in plant reproduction and growth, and also protect against pathogens. The most commonly known flavonoids of phenolic compounds are known to play an important role as secondary metabolites in plants. Representative flavonoids are known to have various biological effects such as antiviral, antimicrobial, anticarcinogenic, anti-inflammatory, antioxidant, antiallergic and antithrombotic effects on animals and humans.

Phenolic acid is abundant in fruit and is divided into benzoic acid and hydroxycinnamic acid according to the structure of hydroxylation and methoxylation in the structure having one phenol ring. Belonging to benzoic acid, gallic acid, protocatechuic acid, syringic acid, p-hydroxybenzoic acid, vanillic acid, and gentisic acid Cinnamic acid, which is abundant in fruits, includes caffeic acid, p-coumaric acid, ferulic acid, chlorogenic acid, cinnamic acid, Sinafic acid. In particular, hydroxycinnamic acid is found in various ways mainly as extraction, treatment, physiological stress, anaerobic conditions or precursors of each substance.

Caffeic acid, p-coumaric acid, and ferulic acid are found in many fruits, and sinafic acid is found in lemons, oranges, pineapples, pumpkins, and tomatoes.

In particular, caffeic acid (3,4-dihydroxycinnamic acid) belongs to phenolic acid, which is mainly ingested in food is rich in fruits, grains, etc., and inhibits 5-, 12-lipoxygenase (5-, 12-lipoxygenase) It is known. 12-lipoxygenase is involved in the metabolism of producing arachidonic acid as 12-hydroperoxyeicosatetraenic acid (12-HPETE) in platelets. HPETE produces 12 (S) -hydroxyeicosatetraenoic acid (12 (S) -hydroxyeicosatetraenoic (12 (S) -HETE), which is involved in platelet aggregation. Therefore, it can be assumed that caffeic acid is involved in the mechanism of antiplatelet action.

On the other hand, platelet action is largely bilateral and physiologically hemostatic action, but pathologically cause blood clots. When blood vessels are wounded and the exposed collagen adheres to platelets circulating in the blood vessels, cellular signaling pathways are activated, resulting in shape changes (morphological changes), secretion and aggregation. Platelet aggregation reactions are induced by stimulants such as collagen, thrombin, and ADP. Among the stimulants, collagen is involved in the early stage of platelet activation and causes platelet aggregation.

When a signal is initiated at a platelet surface receptor, a second messenger such as intracellular Ca ²⁺ is involved in the reaction. [Ca ²⁺ ] _i is increased by inositol 1,4,5-trivalent acid (inositol 1,4,5-trisphosphate, IP ₃ ) being recruited to the cytoplasm of platelets by acting on the IP ₃ receptor of the endoplasmic reticulum. The Ca ²⁺ is combined with calmodulin Ca ²⁺ / calmodulin-dependent phosphorylation (Ca ²⁺ / calmodulin -dependent phosphorylation) deep myosin light chain associated with platelet aggregation (myosin light chain, 20 kDa) to participate in The protein kinase C (PKC) -dependent phosphorylation also phosphorylates the cellular protein pleckstrin (47 kDa). In addition, arachidonic acid, which is degraded and produced from diglycerides, is a precursor of thromboxane A ₂ (TXA ₂ ) that aggregates platelets, which increases the concentration of [Ca ²⁺ ] _i . TXA ₂ itself induces platelet secretion and morphological changes, activates platelets, simultaneously raises the concentration of [Ca ²⁺ ] _i and contributes to phosphorylation of myosin light chain and pleckstrin. Activation of platelets is a cause of cerebrovascular, cardiovascular and peripheral vascular diseases and the treatment of these coronary artery diseases is an important situation. Therefore, inhibiting platelet adhesion and aggregation is the basis for preventing blood clots, and aspirin, which is widely known as an anti-platelet drug, is known to inhibit the formation of TXA ₂ by inhibiting cyclooxygenase. have. Such aspirin is used to prevent angina, ischemic paralysis, stroke, and atherosclerosis in addition to platelet aggregation.

The inventors of the present invention revealed the specific platelet aggregation inhibitory action and mechanism of caffeic acid and chlorogenic acid, and in particular, the antithrombotic effect is excellent in promoting the production of cAMP and cGMP, which lowers the concentration of Ca ^{2+, which} is related to platelet aggregation. The effect was found to be excellent.

Therefore, an object of the present invention is to provide an antiplatelet agent which can effectively prevent the production of blood clots.

In order to achieve the above object, the present invention provides an antiplatelet agent containing at least one of caffeic acid and chlorogenic acid to promote cAMP or cGMP production.

The antiplatelet composition according to the present invention can provide excellent antithrombotic action by increasing the production of cAMP and cGMP by containing caffeic acid or chlorogenic acid, which can be more effective than aspirin known to have antithrombotic action.

The antithrombotic agent of the present invention contains at least one of caffeic acid and chlorogenic acid.

Caffeic acid (3,4-Dihydroxycinnamic acid) used in the present invention has a molecular weight of 180.16, the molecular formula is (HO) ₂ C ₆ H ₃ CH = CHCO ₂ H, it is represented by the following formula (1).

In addition, chlorogenic acid (1,3,4,5-Tetrahydroxycyclohexanecarboxylic acid 3- (3,4-dihydroxycinnamate) used in the present invention has a molecular weight of 354.31, a molecular formula of C ₁₆ H ₁₈ O ₉ , and is represented by the following Chemical Formula 2: do.

The antiplatelet composition according to the present invention is applicable in conventional amounts in the art, and is not particularly limited with respect to the amount.

The antithrombotic composition according to the present invention provides excellent antiplatelet function by increasing the amount of platelet aggregation inhibitory factors cAMP (cyclic adenosine monophosphate) and cGMP (cyclic-guanosine monophosphate).

Hereinafter, the configuration and effects of the present invention will be described in more detail with reference to test examples and examples. However, these test examples and experimental examples are provided only for the purpose of illustration to help the understanding of the present invention is not limited to the scope and scope of the present invention.

Reference Example Preparation of Sample

Caffeic acid and chlorogenic acid were dissolved in 0.1% dimethyl sulfoxide (DMSO), and caffeic acid, chlorogenic acid and DMSO were purchased from Sigma Chemical Co., St. Louis, Missouri, USA.

Collagen was also purchased from Chrono-Log Corporation (Havertown, Pennsylvania, USA), and cAMP- and cGMP-EIA kits were purchased from Amersham bioscience (UK). One was used.

Test Example 1 Measurement of Collagen Concentration Suitable for Rat Platelet Aggregation

1. Preparation of washed rat platelets

Rat platelet-rich plasma (PRP) was centrifuged at 220 x g for 10 minutes to precipitate extra cells, ie, white blood cells and red blood cells, to obtain pure PRP only. This was again centrifuged at 1650 × g for 10 minutes to separate platelet-poor plasma (PPP) and platelet pellet (pellets, PLTs) layers. PLTs were carefully washed 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, and 1 mM EDTA, pH 6.5). After sufficient washing, the precipitated PLTs were separated by centrifugation again at 1650 × g for 10 minutes, and then suspended suspension (138 mM NaCl, 2.7 mM KCl, 12 mM NaHCO ₃ , 0.36 mM NaH ₂ PO ₄ , 0.49 mM). MgCl ₂ , 5.5 mM glucose, 0.25% gelatin, pH 6.9) was added to prepare a cell suspension, and then the suspension was added in an appropriate amount to adjust the cell number to 5 × 10 ⁸ platelets / ml.

2. Collagen Concentration Suitable for Rat Platelet Aggregation

In order to determine the appropriate collagen concentration in the platelet aggregation reaction of collagen-induced mice, platelets (10 ⁸ platelets / ml) prepared in the above manner were pretreated at 37 ° C. for 2 minutes in the presence of 2 mM CaCl ₂ , and then platelets of mice Were stimulated with various concentrations of collagen (0.5-20 μg / ml). The platelet aggregation rate (%) according to the collagen concentration was determined by measuring the light transmittance at 660 nm of the coagulation analyzer (Lumiaggregometer, Chrono-Log, Havertown, PA), and the data were average ± standard deviation (n = 3). Expressed.

Specific methods for calculating the platelet aggregation rate (%) from the light transmittance are as follows.

The transmittance of light is up to 100%, and the platelet aggregation rate (%) increases as the transmittance increases. As shown in Fig. 1, the recording paper of the aggregation system is composed of 0% to 100% scale. When light penetrates the platelet-containing suspended platelet suspension to 10% of this recording paper, and is fixed to 0 as the base line, and the light penetrates the platelet-free washing buffer. Is set to 90%, fixed at the maximum transmittance, and then platelet aggregation reaction is performed by adding collagen to the suspended platelets of the washed platelets. As this increases, the aggregation reaction curve is drawn on the recording sheet. Therefore, the increase in permeability from the 10% point of the washed platelet here means an increase in the aggregation reaction rate.

However, since the permeability increased by collagen is measured in a fixed state by converting 100% permeability to 80% (90%-10% = 80%) in the recording paper of the aggregation system, the actual platelet aggregation rate (%) is expressed by the following equation. Is calculated as 1.

Maximum Platelet Aggregation (%) = A / 80 x 100

(Where A is the transmittance after 5 minutes of collagen addition to the washed platelet suspension prepared from blood of rats with or without drug)

The results for platelet aggregation rates obtained by the above method are shown in FIG. 2.

As can be seen in Figure 2, the platelet aggregation rate and collagen concentration shows a proportional relationship until the concentration of collagen is 10㎍ / ml, but at the collagen concentration of 10 ~ 20㎍ / ml does not change the light transmittance It can be seen that there is almost no change in the aggregation rate of platelets.

According to the above results, the following test was conducted with stimulation of collagen 10 µg / ml showing the highest aggregation rate.

[Test Example 2] Measurement of Platelet Aggregation Inhibition in Rats by Collagen Attraction of Caffeic Acid and Chlorogenic Acid

The washed rat platelet (10 ⁸ platelets / ml) of Test Example 1 in an in vitro reaction system was pre-incubated at 37 ° C. for 2 minutes in the presence of 2 mM CaCl _2. , The treatment of caffeine or chlorogenic acid in platelets at concentrations of 10, 30 and 50μM to examine the aggregation rate of the platelets, the results are shown in Figures 3a and 4a, respectively.

In addition, collagen (10 µg / ml), which is a stimulant, was added to the pre-cultured platelets to cause platelet aggregation reaction for 5 minutes, which was used as a control. In the same manner, caffeic acid or chlorogenic acid was added for each concentration, followed by pretreatment for 2 minutes, followed by stimulation with collagen (10 µg / ml) for platelet aggregation for 5 minutes, and the results are shown in FIGS. 3B and 4B, respectively.

When platelet aggregation occurs due to stimulation of collagen, light transmittance increases. Therefore, platelet aggregation measurement is performed using an aggregometer (aggregometer, Chrono-Log Corp., Havertown, PA). Measured at nm, data are expressed as mean ± standard deviation (n = 3).

Referring to FIG. 3A, when caffeine acid was stimulated on platelets (10 ⁸ platelets / ml) at concentrations of 10, 30, and 50 μM, platelet aggregation rates were 4% at 10 μM, 5% at 30 μM, and 4% at 50 μM, respectively. This is not significantly different from the 5% agglomeration rate obtained in the platelets without caffeic acid, so that if the stimulation with collagen, even if pretreated with caffeic acid, platelet aggregation is hardly achieved.

On the other hand, in Figure 3b, pretreatment of caffeic acid in rat platelets (10 ⁸ platelets / ml) after stimulation at a concentration of collagen 10㎍ / ml, it can be seen that the aggregation is suppressed depending on the concentration of caffeic acid.

In addition, in FIG. 4A, when chlorogenic acid was stimulated to platelets (10 ⁸ platelets / ml) at concentrations of 10, 30, and 50 μM, platelet aggregation rates were 3% at 10 μM, 3% at 30 μM, and 4% at 50 μM. This is not significantly different from the 4% aggregation rate of the platelets without the addition of chlorogenic acid, and when the stimulation with collagen, it can be seen that the platelet aggregation is hardly achieved even if pretreated with chlorogenic acid.

On the other hand, in Figure 4b, pretreatment with chlorogenic acid in rat platelets (10 ⁸ platelets / ml) and then stimulated with a concentration of collagen 10㎍ / ml, it can be seen that the aggregation is suppressed depending on the concentration of chlorogenic acid.

Therefore, it can be seen from the above results that both caffeic acid and chlorogenic acid inhibit platelet aggregation in mice attracted to collagen, and platelet aggregation inhibition is dependent on the concentration of caffeic acid or chlorogenic acid.

Test Example 3 Measurement of cAMP and cGMP Production Effects of Caffeic Acid and Chlorogenic Acid

In general, cAMP and cGMP is known to have an effect of inhibiting platelet aggregation by lowering Ca ²⁺ concentration which is deeply related to platelet aggregation. Therefore, cAMP and cGMP, which are platelet aggregation inhibitors, were measured.

The platelets (10 ⁸ platelets / ml) of the rat prepared in Test Example 1 were treated with various concentrations (10, 30 and 50 μM) of caffeic acid and chlorogenic acid in the presence of 2 mM CaCl ² beforehand at 37 ° C. for 2 minutes. Stimulation with collagen (10 μg / ml). After the reaction occurred, the reaction was stopped by adding 80% ethanol. The production of cAMP and cGMP was measured by cAMP and cGMP EIA kit (Amersham Pharmacia Biotech), the results of the treatment of caffeine is shown in Figure 5a (cAMP production) and 5b (cGMP production), chlorogenic acid is the result of Figure 6a (cAMP Production amount) and 6b (cGMP production amount).

Data are expressed as mean ± standard deviation (n = 3).

5A and 6A, the amount of cAMP produced was decreased in platelets (3.56 pmol / 10 ⁸ platelets) to which collagen (10 μg / ml) was added, compared to platelets (3.76 pmol / 10 ⁸ platelets) to which no stimulation was applied. Alternatively, in the platelets pretreated with chlorogenic acid, cAMP production was significantly increased depending on the concentration.

5B and 6B, as in the case of cAMP, the amount of cGMP produced by platelets (1.09 pmol / 10 ⁸ platelets) to which collagen (10 μg / ml) was added was higher than that of the platelets (1.24 pmol / 10 ⁸ platelets) without stimulation. Although reduced, platelet pretreated with caffeic acid or chlorogenic acid can be seen to significantly increase the amount of cGMP produced depending on the concentration.

1 shows a recording sheet of a cohesive system, which is composed of a scale of 0% to 100%.

2 is a graph showing the aggregation rate (%) of the collagen according to the concentration of collagen.

Figure 3a shows the platelet aggregation rate of mice when not treated with collagen after pretreatment with caffeic acid, Figure 3b shows the platelet aggregation rate of mice when stimulated with collagen after pretreatment with caffeic acid (collagen) When compared with the aggregation rate when stimulated by using alone, *: P <0.05, **: P <0.001).

Figure 4a shows the platelet aggregation rate of mice when not treated with collagen after pretreatment with chlorogenic acid, Figure 4b shows the platelet aggregation rate of mice when stimulated with collagen after pretreatment with chlorogenic acid (collagen alone *: P <0.05, **: P <0.001) compared with the aggregation rate when stimulated with.

Figure 5a shows the amount of cAMP produced after the treatment of caffeic acid, Figure 5b shows the amount of cGMP generated after the treatment of caffeic acid (in comparison with the aggregation rate when stimulated using collagen alone, *: P <0.05, **: P <0.001).

Figure 6a shows the amount of cAMP production after treatment with chlorogenic acid, Figure 6b shows the amount of cGMP production after treatment with chlorogenic acid (compared to the aggregation rate when stimulated using collagen alone, *: P < 0.05, **: P <0.001).

Claims (2)

An antiplatelet agent containing at least one of caffeic acid and chlorogenic acid to increase the amount of cAMP (cyclic adenosine monophosphate). An antiplatelet agent containing at least one of caffeic acid and chlorogenic acid to increase the production of cyclic-guanosine monophosphate (cGMP).

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