KR20220020476A - Composition comprising steppogenin for prevention or treatment of thrombotic disorders - Google Patents

Abstract

The present invention relates to a composition for prevention or treatment of thrombotic diseases containing steppogenin as an active component, and health functional food for prevention or alleviation of thrombotic diseases. More specifically, the present invention is to provide a composition for preventing or treating thrombotic diseases, and health functional food for prevention or alleviation of thrombotic diseases, by revealing an antiplatelet effect on steppogenin, which is a tree root extract of Cudrania tricuspidata.

Description

Composition comprising steppogenin for prevention or treatment of thrombotic disorders, and a composition comprising steppogenin for prevention or treatment of thrombotic disorders

The present invention relates to a composition for the prevention or treatment of thrombotic diseases containing Steppogenin as an active ingredient, and to a health functional food for prevention or improvement. In more detail, by revealing the anti-platelet effect on Steppogenin, a tree root extract of Cudrania tricuspidata, it is used as a composition for preventing or treating thrombotic diseases and as a health functional food for prevention or improvement. in order to be able to provide it.

Since thrombus plays a central role in the occurrence and progression of vascular disease in the circulatory system, it is important to prevent the formation of thrombus and effectively remove the thrombus in the treatment of vascular disease. Thrombosis is a pathologically corresponding concept to the normal hemostasis process, and may occur when platelet aggregation and plasma coagulation processes are excessively activated. Drugs for the prevention and treatment of thrombotic diseases include thrombolytic agents, anticoagulants and antiplatelet agents. Anticoagulants such as heparin and coumarin, which block the blood coagulation system to inhibit the formation of blood clots, and thrombolytic agents that dissolve fibrin, a major component of blood clots, to dissolve already formed clots are used for emergency treatment of thrombotic diseases, but vascular damage It is not suitable for long-term treatment due to side effects such as local bleeding or systemic bleeding.

On the other hand, Cudrania tricuspidata, which belongs to the Morus family and is also called Gutthorn, is a native plant in Korea and is widely distributed in Chungcheong-do, Jeolla-do, and Gyeongsang-do. The extract of Cudrania biloba has been used in folk medicine throughout East Asia in China, Korea, and Japan, where it has been used to treat eczema, mumps, tuberculosis, bruises, insomnia, and acute arthritis. In Donguibogam, there are records that it is good for nourishment and tonicity and yin, physical weakness, insomnia, etc., and its stems and roots are good for female diseases. In Korean folklore, it is known that Cuji mulberry tree decoction with water is effective in treating liver cancer. Cudrania has thorns, and leaves are long oval-shaped, 6-10 cm long and 3-6 cm wide, with fine hairs on the surface and cilia on the back. The fruits are round, and the ripe fruits are red when fully ripened. When the fruits are picked, a white, thick liquid is released, which is known to be free from pests and diseases. It is also known to improve kidney function and intestinal metabolism, thereby improving constipation. In the present invention, it is intended to find out the anti-platelet effect on Stepogenin, an extract of Cudrania biloba, and use it as a composition for preventing or treating thrombotic diseases.

Korean Patent Registration No. 10-2020709 (2019.9.10.) Korean Patent Registration No. 10-1672138 (2016.11.03.)

Kramer, R. M., Roberts, E. F., Um, S. L., Brsch-Haubold, A. G., Watson, S. P., Fisher, M. J. and Jakubowski, J. A. (1996) p38 mitogen-activated protein kinase phosphorylates cytosolic phospholipase A2 (cPLA2) in thrombin-stimulated platelets evidence that proline-directed phosphorylation is not required for mobilization of arachidonic acid by cPLA2. J. Biol. Chem. 271: 27723-27729. McNicol, A. and Shibou, T. S. (1998) Translocation and phosphorylation of cytosolic phospholipase A2 in activated platelets. Thromb. Res. 92: 19-26. Ruggeri, Z. M. and Mendolicchio, G. L. (2007) Adhesion mechanisms in platelet function. Circ. Res. 100: 1673-1685.

Laurent, V., Loisel, T. P., Harbeck, B., Wehman, A., Grbe, L., Jockusch, B. M. and Carlier, M. F. (1999) Role of proteins of the Ena/VASP family in actin-based motility of Listeria monocytogenes. J. Cell. Biol. 144: 1245-1258. Sudo, T. and Ito, H., Kimura, Y. (2003) Phosphorylation of the vasodilator-stimulated phosphoprotein (VASP) by the anti-platelet drug, cilostazol, in platelets. Platelets 14: 381-390. Kuo, J. F., Andersson, R. G., Wise, B. C., Mackerlova, L., Salomonsson, I. and Brackett, N. L. (1980) Calcium-dependent protein kinase: widespread occurrence in various tissues and phyla of the animal kingdom and comparison of effects of phospholipid, calmodulin, and trifluoperazine. Proc. Natl. Acad. Sci. USA. 77: 7039-7043. Nishikawa, M., Tanaka, T. and Hidaka, H. (1980) Ca2+ -calmodulin-dependent phosphorylation and platelet secretion. Nature 287: 863-865. Berridge, MJ and Irvine, RF (1984) Inositol trisphosphate, a novel second messenger in cellular signal transduction. Nature 312: 315-321. Phillips, DR, Nannizzi-Alaimo, L. and Prasad, KS (2001) Beta3 tyrosine phosphorylation in alphaIIb beta3 (platelet membrane GPIIb-IIIa) outside-in integrin signaling. Thromb. Haemost. 86: 246-258. Estevez, B., Shen, B. and Du, X. (2015) Targeting integrin and integrin signaling in treating thrombosis. Arterioscler. Thromb. Vasc. Biol. 35: 24-29. McNicol, A. and Shibou, TS (1998) Translocation and phosphorylation of cytosolic phospholipase A2 in activated platelets. Thromb. Res. 92: 19-26. Needleman, P., Moncada, S., Bunting, S., Vane, JR, Hamberg, M. and Samuelsson, B. (1976) Identification of an enzyme in platelet microsomes which generates thromboxane A2 from prostaglandin endoperoxides. Nature 261: 558-56 Patrignani, P., Sciulli, MG, Manarini, S., Santini, G., Cerletti, C. and Evangelista, V. (1999) COX-2 is not involved in thromboxane biosynthesis by activated human platelets. J. Physiol. Pharmacol. 50: 661-667. Hantgan, RR, Taylor, RG and Lewis, JC (1985) Platelets interact with fibrin only after activation. Blood 65: 1299-1311. Schwarz, U., R., Walter, U. and Eigenthaler, M. (2001) Taming platelets with cyclic nucleotides. Biochem. Pharmacol. 62: 1153-1161. Chin, HS and Nam, KW (2010) Inhibitory Effects of Steppogenin and Oxyresveratrol from Moms alba L. against Yeast a-Glucosidase. Yakhak Hoeji 54: 398-402. Kim, DC, Quang, TH, Oh, H. and Kim, YC (2017) Steppogenin isolated from cudrania tricuspidata shows antineuroinflammatory effects via NF-κB and MAPK pathways in lps-stimulated bv2 and primary rat microglial cells. Molecules 22: 2130; doi:10.3390/molecules22122130. Grynkiewicz, G., Poenie, M. and Tsien, RY (1985) A new generation of Ca2+ indicators with greatly improved fluorescence properties. J. Biol. Chem. 260: 3440-3450. Xin, LT, Yue, SJ, Fan, YC, Wu, JS, Yan, D., Guan, HS and Wang, CY (2017) Cudrania tricuspidata: an updated review on ethnomedicine, phytochemistry and pharmacology. RSC advances 7: 31807-31832. Ro, JY and Cho, HJ (2019) Cudrania Tricuspidata root extract (CTE) has an anti-platelet effect via cGMP-dependent VASP phosphorylation in human platelets. J. Korea. Acad. Industry. Coop. Soc. 20: 298-305. Varga-Szabo, D., Braun, A. and Nieswandt, B. (2009). Calcium signaling in platelets. J. Thromb. Haemost. 7: 1057-1066. Kwon, HW (2018) Inhibitory effects of PD98059, SB203580, and SP600125 on α-and δ-granule release and intracellular Ca2+levels in human platelets. Biomed. Sci. Lett. 24: 253-262.

Kramer, RM, Roberts, EF, Um, SL, Brsch-Haubold, AG, Watson, SP, Fisher, MJ and Jakubowski, JA (1996) p38 mitogen-activated protein kinase phosphorylates cytosolic phospholipase A2 (cPLA2) in thrombin-stimulated platelets evidence that proline-directed phosphorylation is not required for mobilization of arachidonic acid by cPLA2. J. Biol. Chem. 271: 27723-27729. McNicol, A. and Shibou, TS (1998) Translocation and phosphorylation of cytosolic phospholipase A2 in activated platelets. Thromb. Res. 92: 19-26. Ruggeri, ZM and Mendolicchio, GL (2007) Adhesion mechanisms in platelet function. Circ. Res. 100: 1673-1685.

Laurent, V., Loisel, TP, Harbeck, B., Wehman, A., Grbe, L., Jockusch, BM and Carlier, MF (1999) Role of proteins of the Ena/VASP family in actin-based motility of Listeria monocytogenes. J. Cell. Biol. 144: 1245-1258. Sudo, T. and Ito, H., Kimura, Y. (2003) Phosphorylation of the vasodilator-stimulated phosphoprotein (VASP) by the anti-platelet drug, cilostazol, in platelets. Platelets 14: 381-390. Kuo, JF, Andersson, RG, Wise, BC, Mackerlova, L., Salomonsson, I. and Brackett, NL (1980) Calcium-dependent protein kinase: widespread occurrence in various tissues and phyla of the animal kingdom and comparison of effects of phospholipids, calmodulin, and trifluoperazine. Proc. Natl. Acad. Sci. USA. 77: 7039-7043.

An object of the present invention is to reveal the antithrombotic effect of steppogenin, an extract of Cudrania tricuspidata, so that steppogenin is used for the prevention, treatment or improvement of thrombotic diseases.

It is an object of the present invention to provide a pharmaceutical composition for the prevention or treatment of thrombotic diseases containing Stepogenin, which is an extract of Cuji mulberry tree, as an active ingredient.

Another object of the present invention is to provide a health functional food for the prevention or improvement of thrombotic diseases containing Stepogenin, which is an extract of Cuji mulberry tree, as an active ingredient.

The technical problems to be achieved by the present invention are not limited to the technical problems mentioned above, and other technical problems not mentioned can be clearly understood by those of ordinary skill in the art to which the present invention belongs from the description below. There will be.

The present invention, devised to achieve the above object, is preferably a composition for preventing or treating thrombotic diseases containing Steppogenin as an active ingredient. In addition, the stepogenin is It is more preferable that it is characterized in that it is a Cuji mulberry tree extract. And the concentration of the kkujippong tree extract is also preferably characterized in that 500 μg / mL. In addition, the Cujippong tree extract is most preferably characterized in that the Cujippong tree root extract.

In addition, the present invention is also preferably as a health functional food for the prevention or improvement of thrombotic diseases containing steppogenin as an active ingredient, wherein the steppogenin is It is more preferable that it is characterized in that it is a cucurbita tree or cucurbita root extract.

Stepogenin, which is an extract of Cuji mulberry tree root according to the present invention, can effectively inhibit platelet activation and thrombus formation, so it has an effect that can be usefully used to prevent and treat various thrombotic diseases caused by thrombosis and platelet aggregation.

Figure 1 shows the chemical structure of stepogenin.
2 is a graph showing the effect of stepogenin on platelet aggregation and cytotoxicity.
Figure 3 is a graph showing the effect of stepogenin on [Ca ^{2 +} ] _i mobilization, IP ₃ RI and ERK phosphorylation.
4 is a graph showing the effect of stepogenin on TXA ₂ production and cPLA ₂ phosphorylation.
5 is a graph showing the effect of steppogenin on fibronectin adhesion and VASP phosphorylation.
Figure 6 shows the effect of stepogenin on cyclic nucleotides.

Hereinafter, the present invention will be described in detail so that the above-described objects and features become clear, and accordingly, those of ordinary skill in the art to which the present invention pertains will be able to easily implement the technical idea of the present invention. In addition, in the description of the present invention, if it is determined that the detailed description of the known technology may unnecessarily obscure the gist of the present invention as it is already well known in the technical field among the known technologies related to the present invention, the detailed description thereof is omitted. to be omitted.

In addition, the terms used in the present invention have been selected as widely used general terms as possible, but in certain cases, there are also terms arbitrarily selected by the applicant, and in this case, the meaning is described in detail in the description of the corresponding invention, so it is a simple term It is intended to clarify that the present invention should be understood as the meaning of the term, not the name. Terms used in the description of the embodiments are only used to describe specific embodiments, and are not intended to limit the embodiments. The singular expression includes the plural expression unless the context clearly dictates otherwise.

Embodiments may be modified in various forms and may have various additional embodiments, wherein specific embodiments are shown in the drawings and related detailed descriptions are given. However, this is not intended to limit the embodiments to specific forms, and should be understood to include all changes or equivalents or substitutes included in the spirit and scope of the embodiments.

Hereinafter, the present invention will be described. It is well known that platelets are essential cells for hemostasis of blood vessel damage and can act as risk factors in patients at risk for cardiovascular disease. Collagen exposed to damaged blood vessels activates circulating platelets, and phosphatidylinositol 4,5-bisphosphate in the activated platelet membrane is hydrolyzed into inositol-1,4,5-triphosphate (IP ₃ ) and diacylglycerol. The generated IP ₃ binds to a receptor (IP3 receptor type I) present on the endoplasmic reticulum surface inside the cytoplasm of platelets ^{and releases stored Ca 2+ into} the cytoplasm. ^{Increased Ca 2+ in} platelets causes platelet shape change and granule release through the inside-out signaling pathway, and activates the platelet membrane glycoprotein IIb/IIIa (αIIb/β3) to trigger the outside-in signaling pathway to activate platelets. induce When the inside-out signaling pathway starts, cytosolic phospholipase A ₂ , an enzyme present in the platelet cytoplasm, is activated in the presence of Ca ^{2+ and} moves to the platelet membrane, hydrolyzes fatty acids in phospholipids, and liberates arachidonic acid into the cytoplasm make it After that, arachidonic acid is synthesized as thromboxane A ₂ by the action of an enzyme and released to the outside of platelets. Platelets activated through various actions create a thrombus and complete the hemostasis.

Cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) are well known as platelet inhibitory molecules generated in the human body. During normal blood circulation, vascular endothelial cells release nitric oxide and prostaglandin I ₂ to increase the concentration of cAMP and cGMP inside platelets. Increased cAMP induces activation of protein kinase A (PKA), whereas an increase in cGMP induces activation of protein kinase G (PKG). The substrates of PKA and PKG are inositol 1,4,5-triphosphate receptor type I (IP ₃ RI) and vasodilator-stimulated phosphoprotein (VASP), both of which act as inhibitory molecules that inhibit platelet activation when phosphorylated.

Steppogenin found in Cudrania tricuspidata has been reported to have antineuroinflammatory, anti-tyrosinase, and anti-α-glucosidase actions. However, the function and role of steppogenin in human platelet aggregation is not known so far. Therefore, the inventor of the present invention confirmed various inhibitory effects and mechanisms using collagen-induced human platelets to clearly reveal the antiplatelet effect of steppogenin in the course of carrying out the invention. Hereinafter, the present invention will be described in more detail through examples and experiments.

1. Materials and Methods

go. test material

Steppogenin was purchased from ChemFaces (Wuhan, China), collagen and thrombin were from Chrono-Log (Havertown, PA, USA), Lactate dehydrogenase cytotoxicity assay kit, U46619, cyclic adenosine monophosphate kit and cyclic guanosine monophosphate kit were from Cayman Chemical (Ann Arbor, MI, USA). And other reagents were purchased from Sigma Aldrich (Saint Louis, MO, USA), and the CytoSelect 48-well cell adhesion assay kit was purchased from Cell Biolabs (San Diego, CA, USA). Antibodies for Western blotting and lysis buffer were purchased from Cell Signaling (Beverly, MA, USA), and polyvinylidene difluoride (PVDF) membrane and enhanced chemiluminesence solution (ECL) were purchased from GE Healthcare (Buckinghamshire, UK).

me. human lavage platelets

Anticoagulated human platelet-rich plasma (PRP) with acid-citrate-dextrose solution (0.8% citric acid, 2.2% sodium citrate, 2.45% glucose) was provided from the Korean Red Cross Blood Center (Suwon, Korea). To remove trace amounts of red blood cells, PRP was centrifuged at 125xg for 10 minutes, and then centrifuged at 1,300xg for 10 minutes to obtain platelet pellets. Wash it twice 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). Washed platelets were reconstituted with suspension buffer (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) to a final 108/mL concentration was made. All of the above procedures were performed at 25°C to avoid platelet aggregation that may occur at low temperatures. This experiment was performed with the approval of The Korea National Institute for Bioethics Policy Public Institutional Review Board (Seoul, Korea) (P01-201812-31-007).

all. Measurement of platelet aggregation

Washed platelets (2.5x10 ⁸ /mL) were pretreated with steppogenin (50, 100, 150, 200 μM) at various concentrations at 37°C for 3 minutes, then aggregation was induced with 2.5 μg/mL collagen and measured for 5 minutes. did Aggregation was measured with an aggregometer at 1,000 rpm stirring speed (Chrono-Log, Havertown, PA, USA), and the aggregation capacity was calculated as an increased degree of light transmittance. Suspension buffer was used as a standard value of transmittance of 0%, and steppogenin was dissolved in dimethyl sulfoxide (DMSO) to a final concentration of 0.1%.

La. Cytotoxicity evaluation

Wash platelets (2.5x108/mL) with steppogenin (50, 100, 150, 200 μM) of various concentrations added and pretreated at 37°C for 5 minutes, then centrifuged at 12,000xg for 15 minutes to remove the cell debris. The lactate dehydrogenase (LDH) cytotoxicity assay kit (Cayman Chemical) was used. The value of completely lysing platelets with 0.1% Triton X-100 was set as 100% as a positive control, and the value of steppogenin was presented as %.

mind. Measurement of intracellular Ca ^{2+ mobilization}

Washed platelets (2.5x108/mL) were treated with 5 μM of Fura 2-AM and pre-treated at 37°C for 60 minutes. Then, centrifuged at 1,300xg for 10 minutes and resuspended in suspending buffer to prepare platelets for Ca ^{2 +} mobilization measurement. Washed platelets were pretreated with steppogenin (50, 100, 150, 200 μM) of various concentrations at 37°C for 3 minutes, then aggregation was induced with 2.5 μg/mL collagen and reacted for 5 minutes. The fluorescence wavelength was analyzed at excitation 340 nm and emission 510 nm, and was measured with a spectrofluorometer (Hitachi F-2500, Tokyo, Japan) using Grynkiewicz's method.

bar. Thromboxane A2 measurement

Washed platelets (2.5x108/mL) were pretreated with steppogenin (50, 100, 150, 200 μM) of various concentrations at 37°C for 3 minutes, then aggregation was induced with 2.5 μg/mL collagen and agglutination reaction for 5 minutes was performed. After that, TXA2 synthesis was stopped by treatment with 250 μL ice-cold 5 mM EDTA and 0.2 mM indomethacin. Thereafter, TXB2, a stable metabolite of TXA2, was measured using the TXB2 EIA kit (Cayman Chemical).

buy. Fibronectin Adhesion Measurement

Wash platelets (2.5x108/mL) and various concentrations of steppogenin (50, 100, 150, 200 μM) were added to fibronectin-coated wells, stimulated with 2.5 μg/mL collagen, and pretreated at 37°C for 60 minutes. Fibronectin adhesion was measured. After that, it was washed 5 times with PBS, stained with a stain solution, and extracted using an extract solution. Then, the extract was transferred to a 96-well microtiter plate and analyzed using an ELISA reader (TECAN, Salzburg, Austria).

Ah. Phosphorylation Analysis Using Western Blot

Washed platelets (2.5x108/mL) were pretreated with steppogenin (50, 100, 150, 200 μM) at various concentrations at 37°C for 3 minutes, then aggregation was induced with 2.5 μg/mL collagen and reacted for 5 minutes. . After that, the reaction was stopped by adding the same amount of lysis buffer. For platelet lysate, protein was quantified using BCA protein assay kit (Pierce Biotechnology, IL, USA), and the same amount of protein (15 μg) was used for analysis. For electrophoresis, 8% SDS-PAGE was used, and the protein was transferred to a PVDF membrane, and the transferred membrane was developed with ECL reagent.

ruler. Cyclicnucleotides measurement

Washed platelets (2.5x108/mL) were pretreated with steppogenin (50, 100, 150, 200 μM) of various concentrations at 37°C for 3 minutes, then aggregation was induced with 2.5 μg/mL collagen and agglutination reaction for 5 minutes was performed. Then, 1M HCl was added to stop the reaction, and an ELISA reader (TECAN, Salzburg, Austria) was analyzed using cAMP and cGMP EIA kit.

car. statistical analysis

All measured experimental results were treated as mean±SEM and analyzed by analysis of variance (ANOVA). If there was a significant difference in the mean between groups, it was compared with the Newman-Keuls method and marked between each group. When p<0.05, it was judged to have a significant meaning.

2. Results and Discussion

go. Effect of Steppogenin on Platelet Aggregation and Cytotoxicity

Cudrania tricuspidata extract has long been reported to have various physiological activities such as anti-inflammatory, antioxidant, and anticancer, and studies on its effect on platelets have also been reported recently. However, the identification of a single component derived from the extract has not yet been reported. Therefore, in this study, the antiplatelet effect of steppogenin (see Fig. 1), a component derived from Cudrania tricuspidata extract, and its inhibitory mechanism were clearly identified. To confirm the platelet inhibitory activity of Steppogenin, collagen, thrombin, and U46619 were used as agonists.

2 is a graph showing the effect of Stepogenin on platelet aggregation and cytotoxicity, FIG. 2A is the effect of Stepogenin on collagen-induced human platelet aggregation, and FIG. 2B is Stepogenin on thrombin-induced human platelet aggregation. Figure 2C shows the effect of stepogenin on U46619-induced human platelet aggregation, and Figure 2D shows the effect of stepogenin on cytotoxicity. Here, platelet aggregation and cytotoxicity were performed as described in the materials and methods described above, and data are expressed as mean ± standard deviation (n = 4). As shown in FIG. 2, the aggregation reactions induced by the three agonists were 96.0%, 96.1%, and 91.3%, respectively, respectively, and as a result of treatment with steppogenin (50, 100, 150, 200 μM), a concentration-dependent inhibition pattern was obtained. was confirmed (Fig. 2A, Fig. 2B, Fig. 2C). Steppogenin had the lowest effect on thrombin-induced platelet aggregation and showed the maximum inhibition rate at a concentration of 300 μM (Fig. 2B). Among the three agonists, Steppogenin had the highest inhibitory efficiency in the collagen-induced platelet aggregation reaction, so the experiment was conducted focusing on the effect on the collagen-induced platelet aggregation reaction. Lactate dehydrogenase (LDH) leakage was performed in order to evaluate the cytotoxicity of Steppogenin. Human platelets were treated with steppogenin (50, 100, 150, 200 μM) to analyze LDH leakage. As a result, it did not show significance (Fig. 2D). The Cudrania tricuspidata extract showed an inhibitory effect on the collagen-induced rat platelet aggregation reaction, and showed an inhibitory rate of 86.5% at a concentration of 500 μg/mL.

me. Effects of Steppogenin on Intracellular Ca ²⁺ Mobilization ^and Phosphorylation of IP ₃ RI and ERK

In the cytoplasm of activated platelets, the concentration of Ca ²⁺ ([Ca ²⁺ ] _i ) increases, ^and [Ca ²⁺ ] _i acts as an essential ^secondary messenger for platelet activity, so ^steppogenin acts as an intracellular [Ca ^{2+ ]} The effect on _i was confirmed. 3 is a graph showing the effect of ^stefogenin on _[ Ca ^{2 +} ] _i mobilization, IP ₃ RI and ERK phosphorylation ^. , FIG. 3B is the effect of stepogenin on collagen-induced IP ₃ RI (Ser ¹⁷⁵⁶ ) phosphorylation, and FIG. 3C is the effect of stepogenin on collagen-induced ERK (1/2) phosphorylation. Here, [Ca ²⁺ ] _i mobilization and Weston blots were performed as described in Materials and Methods above, and data are presented as mean ± standard deviation (n = 4). As shown in FIG. 3 , human platelets stimulated with collagen ( FIG. 3A ) were strongly increased to 640.4±11.2 nM, and treatment with steppogenin (50, 100, 150, 200 μM) showed a concentration-dependent inhibition pattern. The concentration of intracellular Ca ²⁺ is inhibited by phosphorylation of inositol ^- 1,4,5-triphosphate receptor type I (IP ₃ RI) present in the membrane of the endoplasmic reticulum. Therefore, as a result of confirming the effect of steppogenin on phosphorylation of IP ₃ RI, steppogenin increased phosphorylation of the Ser ¹⁷⁵⁶ position of IP ₃ RI ( FIG. 3B ). ERK, one of the mitogen-activated protein kinases, is known to be involved in the influx of extracellular Ca ²⁺ into the cell from human platelets, ^and is phosphorylated by agonist stimulation, thereby contributing to platelet activation. Collagen-stimulated human platelets strongly induced ERK phosphorylation, and as a result of treatment with steppogenin (50, 100, 150, 200 μM), a concentration-dependent inhibition pattern was shown ( FIG. 3C ).

all. Effect of Steppogenin on Release of Thromboxane A ₂ and Phosphorylation of cPLA2

TXA ₂ synthesized inside platelets is released to the outside of platelets and acts as an agonist on the released platelets and circulating platelets. Therefore, the effect on the synthesis of TXA2 was evaluated by treating the collagen-stimulated platelets with steppogenin (50, 100, 150, 200 μM). As a result, steppogenin concentration-dependently inhibited TXA ₂ production increased by collagen (FIG. 4A). Figure 4 is a graph showing the effect of Stepogenin on TXA ₂ production and cPLA ₂ phosphorylation, Figure 4A is the effect of Stepogenin on collagen-induced TXA ₂ production, Figure 4B is collagen-induced cPLA ₂ (Ser ⁵⁰⁵ ) Effect of Stepogenin on Phosphorylation. Here, measurements of TXA ₂ production and Western blot were performed as described in Materials and Methods, and data were expressed as mean ± standard deviation (n = 4). In order to confirm the exact mechanism for the synthesis of TXA ₂ inhibited by Steppogenin, the activity of cPLA ₂ was analyzed. cPLA ₂ exists in the cytoplasm, ^{and after binding with Ca 2+ ,} moves to the cell membrane, and the Ser ⁵⁰⁵ position is phosphorylated to have enzymatic activity. And TXA ₂ is synthesized from arachidonic acid generated through cPLA ₂ action. Therefore, in order to confirm the effect of steppogenin on cPLA ₂ , phosphorylation of cPLA ₂ was confirmed. Collagen-stimulated human platelets phosphorylated the Ser ⁵⁰⁵ position of cPLA ₂ , and steppogenin showed a concentration-dependent inhibition pattern (FIG. 4B). Therefore, it was confirmed that the release of TXA ₂ inhibited by steppogenin is an action through the phosphorylation inhibitory action of cPLA ₂ .

La. Effect of Steppogenin on Fibronectin Adhesion and VASP Phosphorylation

Plasma protein fibronectin binds to αIIb/β3, an integrin of platelet membrane, and causes platelet adhesion. To evaluate the effect of steppogenin on the activity of αIIb/β3, fibronectin adhesion was analyzed. 5 is a graph showing the effect of steppogenin on fibronectin adhesion and VASP phosphorylation, FIG. 5A is the effect of steppogenin on collagen-induced fibronectin adhesion, and FIG. effect, and FIG. 5C is the effect of Stepogenin on collagen-induced VASP (Ser239) phosphorylation. Here, measurements of fibrinogen binding and fibronectin adhesion were performed as described in Materials and Methods, and data were presented as mean ± standard deviation (n = 4). As shown in FIG. 5, collagen stimulation caused adhesion between αIIb/β3 and fibronectin (FIG. 5A), but concentration-dependent inhibitory activity was shown in wells treated with steppogenin (50, 100, 150, 200 μM). (Fig. 5A). As a factor regulating platelet αIIb/β3, vasodilator stimulated phosphoprotein (VASP) is well known. VASP is a protein that helps actin dynamic action in human platelets, leading to the activity of αIIb/β3. However, in VASP, Ser ¹⁵⁷ and Ser ²³⁹ are phosphorylated by cAMP and cGMP, which are cyclic nucleotides, and the activity of αIIb/β3 is lost. Therefore, as a result of analyzing the phosphorylation of VASP by treating human platelets stimulated with collagen with steppogenin (50, 100, 150, 200 μM), it was confirmed that phosphorylation was increased in VASP Ser ¹⁵⁷ and VASP Ser ²³⁹ (Fig. 5B, Fig. 5B, Fig. 5C).

mind. Effect of Steppogenin on Cyclic Nucleotides

Cyclic nucleotides, cAMP and cGMP, are known to activate protein kinase A (PKA) and protein kinase G (PKG), respectively, and phosphorylate their respective substrates to inhibit platelet activity. Representative substrates of PKA and PKG in platelets are well known as IP ₃ RI and VASP, and they act to circulate blood vessels by maintaining platelets in a resting state. In the previous experiment, steppogenin increased the phosphorylation of IP ₃ RI and VASP, so it was thought that it was related to the concentration of cAMP and cGMP, and the concentration of cAMP and cGMP inside the platelets stimulated with steppogenin was analyzed. As a result, it was confirmed that steppogenin increased the concentration of cyclic nucleotides in human platelets stimulated with collagen. Figure 6 shows the effect of stepogenin on cyclic nucleotides, Figure 6A is the effect of stepogenin on cAMP production, Figure 6B shows the effect of stepogenin on cGMP production. Here, measurements of cAMP and cGMP were performed as described in Materials and Methods, and data were presented as mean ± standard deviation (n = 4). As shown in FIG. 6, Steppogenin (50, 100, 150, 200 μM) increased the concentrations of cAMP and cGMP in a concentration-dependent manner ( FIGS. 6A and 6B ), and the concentration of cyclic nucloetides increased by steppogenin was higher in cAMP than in cGMP. increased more strongly. Through these results, it was confirmed that steppogenin has antiplatelet action by increasing cAMP and cGMP inside platelets. Cudrania tricuspidata extract increased the concentration of cGMP in the collagen-induced rat platelet aggregation reaction, but did not increase the concentration of cAMP. The results of Cudrania tricuspidata extract were different from the results of cAMP and cGMP, which steppogenin increased, and it was assumed that this was due to the difference in the action of phosphodiesterase that degrades cyclic nucleotides.

3. Conclusion

Platelet activation leads to inside-out signaling ^{and promotes platelet αIIb/β3 activation along with intracellular Ca 2+ secretion} . Activated αIIb/β3 forms a strong hemostatic stopper by forming a cross-link through the adhesive protein fibronectin. In this study, the effect of steppogenin on platelet aggregation was evaluated. Steppogenin strongly inhibited platelet aggregation induced by collagen, and it was confirmed that these results occurred through the regulation of phosphorylation of IP ₃ RI and VASP, which are related signaling molecules. Therefore, steppogenin is considered to have potential value as a substance that inhibits human platelet activity.

4. Summary of Results

Cudrania tricuspidata extract is used in folk medicine throughout East Asia in China, Korea and Japan. In oriental medicine, mulberry tree has been used to treat eczema, mumps, tuberculosis, bruises, insomnia, and acute arthritis. In the present invention, it was investigated whether any compound has an inhibitory effect on platelet aggregation in Cudrania tricuspidata, that is, Cudrania tricuspidata. Through this invention, we tried to explain the mechanism of inhibition of steppogenin from Cudrania tricuspidata on human platelet aggregation. Collagen-induced human platelet aggregation and [Ca ²⁺ ] _i mobilization were dose-dependently inhibited by steppogenin, ^and steppogenin-induced inhibition of extracellular signal-regulated kinase (ERK) and inositol-1,4,5-triphosphate receptor type I ( IP ₃ RI) was due to downregulation of phosphorylation. In addition, steppogenin inhibited collagen-induced fibronectin adhesion to αIIb/β3 and thromboxane A ₂ production. Therefore, through the present invention, steppogenin has demonstrated the inhibitory effect of human platelet aggregation, which means that it can be used to prevent platelet-induced cardiovascular disease.

Although the present invention has been described with various examples described above, the present invention is not necessarily limited to these examples, and various modifications may be made within the scope without departing from the technical spirit of the present invention. Therefore, the examples disclosed in the present invention are not intended to limit the technical spirit of the present invention, but to explain, and the scope of the technical spirit of the present invention is not limited by these examples. The protection scope of the present invention should be construed by the following claims, and all technical ideas within the scope equivalent thereto should be construed as being included in the scope of the present invention.

Claims (6)

Composition for the prevention or treatment of thrombotic diseases containing Steppogenin as an active ingredient According to claim 1,
The Stepogenin is Composition for the prevention or treatment of thrombotic diseases, characterized in that it is an extract of Cuji mulberry tree 3. The method of claim 2,
The composition for the prevention or treatment of thrombotic diseases, characterized in that the concentration of the Cuji mulberry extract is 500 μg/mL 4. The method of claim 2 or 3,
The Cuji mulberry tree extract is a composition for the prevention or treatment of thrombotic diseases, characterized in that the Cuji mulberry root extract Health functional food for the prevention or improvement of thrombotic diseases containing Steppogenin as an active ingredient 5. The method of claim 4,
The stepogenin is a health functional food for the prevention or improvement of thrombotic diseases, characterized in that the extract of Cuji mulberry tree or Cuji mulberry root extract

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