• Korean Journal of Clinical Laboratory Science
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• v.51 no.1
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• pp.34-41
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• 2019

Platelet aggregation is essential for the formation of a hemostatic plug in the case of blood vessel damage. On the other hand, excessive platelet aggregation may cause cardiovascular disorders, such as thrombosis, atherosclerosis, and myocardial infarction. Scopoletin, which found in the root of plants in the genus Scopolia or Artemisia, has anti-coagulation and anti-malaria effects. This study examined the effects of scopoletin on human platelet aggregation induced by collagen. Scopoletin had anti-platelet effects via the down-regulation of thromboxane $A_2$ ($TXA_2$) production and intracellular $Ca^{2+}$ mobilization ($[Ca^{2+}]_i$), which are aggregation-inducing molecules produced in activated platelets. On the other hand, scopoletin increased both the cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) levels, which are known as intracellular $Ca^{2+}$-antagonists and aggregation-inhibiting molecules. In particular, scopoletin increased the potently cAMP level more than cGMP, which led to suppressed fibrinogen binding to ${\alpha}IIb/{\beta}_3$ in collagen-induced human platelet aggregation. In addition, scopoletin inhibited collagen-elevated adenosine triphosphate (ATP) release in a dose-dependent manner. The results suggest that aggregation amplification through granule secretion is inhibited by scopoletin. Therefore, scopoletin has potent anti-platelet effects and may have potential for the prevention of platelet-derived vascular diseases.

• The Korean Journal of Physiology
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• v.23 no.2
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• pp.263-275
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• 1989

Mechanical contractions and electrical activities of the fundic longitudinal and antral circular muscle fibers were investigated in order to elucidate topical differences of gastric motility. K-induced contracture was produced by exposure of muscle strips to high K Tyrode solution. Membrane potential and mechanical contraction were simultaneously recorded by conventional glass microelectrode method and single sucrose-gap technique. All experiments were performed in tris-buffered Tyrode solution which was aerated with $100%\;O_2\;and\;kept\;35^{\circ}C$. The results obtained were as follows: 1) The resting membrane potential of circular muscle cells in the antral region was about 10 mV more negative than that in the fundic region. 2) The membrane potentials decreased almost linearly as the extracellular KCI concentration was increased both in antral circular muscle cells and in fundic longitudinal muscle cells. 3) The thresholdal K concentration of K-contracture was 15 mM (membrane potential, -48 mV) for the antral circular muscle strip and 20 mM for the fundic longitudinal muscle cells. 4) The ratio of membrane permeability coefficient for $Na^+\;and\;K^+,\;P_{Na}/P_K\;({\alpha})$ was 0.065 for antral circular muscle cells and was 0.108 for fundic longitudinal muscle cells. 5) K-contracture of antral and fundic smooth muscle strips showed the contracture composed of phasic and tonic components. The amplitude of the phasic component increased sigmoidally in a dose-dependent manner, whereas that of the tonic component was maximal at a concentration of 40 mM KCI and at the concentrations above or below 40 mM KCI the amplitude was reduced. 6) The inverse relationship between the amplitude of tonic component and extracellular KCI concentration in the range of 40 to 150 mM KCI was more prominent in the antral circular muscle strip than in the fundic longitudinal muscle strip, where the amplitude of the tonic component decreased less steeply and was maintained higher at the same high K concentrations. 7) The tonic component was totally dependent on the external $Ca^{2+}$ and completely abolished by verapamil, while tile phasic component was far less dependent on the external $Ca^{2+}$ and partially suppressed by verapamil. From the above results, the following conclusions could be made. 1) The phasic component of K-contracture is produced both by intracellular $Ca^{2+}$ mobilization and by $Ca^{2+}$-influx from outside, while the tonic component is generated and maintained by the $Ca^{2+}-influx$ through the potential-dependent $Ca^{2+}$ channel. 2) The mechanism of reducing the free $Ca^{2+}$ concentration in the myoplasm seems to be more developed in the antral circular muscle than in the fundic longitudinal muscle. 3) The lower resting membrane potential of the fundic longitudinal muscle cell reflects a relatively high $P_{Na}/P_K$ ratio of about 0.108.

• Journal of Ginseng Research
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• v.39 no.3
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• pp.279-285
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• 2015

Background: Korean Red Ginseng has been used as a traditional oriental medicine to treat illness and to promote health for several thousand years in Eastern Asia. It is widely accepted that ginseng saponins, ginsenosides, are the major active ingredients responsible for Korean Red Ginseng's therapeutic activity against many kinds of illness. Although the crude saponin fraction (CSF) displayed antiplatelet activity, the molecular mechanism of its action remains to be elucidated. Methods: The platelet aggregation was induced by collagen, the ligand of integrin ${\alpha}_{II}{\beta}_I$ and glycoprotein VI. The crude saponin's effects on granule secretion [e.g., calcium ion mobilization and adenosine triphosphate (ATP) release] were determined. The activation of mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated protein kinase 1/2 (ERK1/2), c-Jun N-terminal kinases (JNKs), and p38 MAPK, and phosphoinositide 3-kinase (PI3K)/Akt was analyzed by immunoblotting. In addition, the activation of integrin ${\alpha}_{II}b{\beta}_{III}$ was examined by fluorocytometry. Results: CSF strongly inhibited collagen-induced platelet aggregation and ATP release in a concentration-dependent manner. It also markedly suppressed $[Ca^{2+}]_i$ mobilization in collagen-stimulated platelets. Immunoblotting assay revealed that CSF significantly suppressed ERK1/2, p38, JNK, PI3K, Akt, and mitogen-activated protein kinase kinase 1/2 phosphorylation. In addition, our fraction strongly inhibited the fibrinogen binding to integrin ${\alpha}_{IIb}{\beta}_3$. Conclusion: Our present data suggest that CSF may have a strong antiplatelet property and it can be considered as a candidate with therapeutic potential for the treatment of cardiovascular disorders involving abnormal platelet function.

• Korean Journal of Plant Tissue Culture
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• v.27 no.1
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• pp.31-37
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• 2000

Proton extrusion by hairy roots of Panax ginseng C.A. Meyer was enhanced by iron-deficiency or single salt solutions of nitrate or ammonium salts. The enhancement effect of iron-deficiency was confined to the apical zones, whereas that of nitrate and ammonium was confined to the basal root zones. tinder iron-deficiency medium, the proton extrusion of ginseng hairy roots was higher at two times than that grown in the medium of iron-sufficiency. According to pH in the medium, uptake of inorganic elements showed various patterns for the kind of inorganic elements. $Ca^{2＋}$, $Mg^{2＋}$ and Fe$^{2＋}$ were highest uptake at the pH 4.8 and Mn$^{2＋}$, Zn$^{2＋}$ and Cu$^{2＋}$ were highest uptake at the pH 3.8. Free sugars and ginsenosides were analyzed by HPLC. Five kinds of free sugars were indentified in ginseng hairy roots. Glucose, fructose and sucrose were a major sugar in ginseng hairy roots and contents of free sugars were highest at pH 6.8 and pH 4.8, respectively. The contents of ginsenosides and phenolic compounds were highest at pH 5.8. This physiological responses in ginseng hairy root indicated that it may well be a good model system for the study of mechanism of mobilization and uptake of inorganic elements related with the red-colored phenomenon in ginseng roots.

• Journal of Applied Biological Chemistry
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• v.63 no.4
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• pp.339-345
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• 2020

Euchrestaflavanone A (EFA) is a flavonoid found in the root bark of Cudrania tricuspidata. C. tricuspidata extract, widely used throughout Asia in traditional medicine, has been investigated phytochemically and biologically and is known to have anti-obesity, anti-inflammatory, and anti-tumor effects. It has been reported that C. tricuspidata extract also possesses anti-platelet effects; however, the mechanism of its anti-platelet and anti-thrombotic activities is yet to be elucidated. In this study, we investigated the effects of EFA on the modulation of platelet function using collagen-induced human platelets. Our results showed that EFA markedly inhibited platelet aggregation. Furthermore, it downregulated glycoprotein IIb/IIIa (αIIb/β3)-mediated signaling events, including platelet adhesion, granule secretion, thromboxane A2 production, and clot retraction, but upregulated the cyclic adenosine monophosphate-dependent pathway. Taken together, EFA possesses strong anti-platelet and anti-thrombotic properties and is a potential therapeutic drug candidate to prevent platelet-related thrombosis and cardiovascular disease.

• Journal of Physiology & Pathology in Korean Medicine
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• v.27 no.5
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• pp.602-610
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• 2013

This study was aimed to evaluate the cavernosal relaxation effect of Crataegii fructus(CF) in the contracted rabbit penile corpus cavernosum by agonists.In order to study the effect of CF on the vasoconstriction of rabbit penile corpus cavernosum, isolated rabbit penile corpus cavernosum tissues were used for the experiment using organ baths containing Krebs solution.To investigate the cavernosal relaxation of CF, CF extract at $0.01{\sim}3.0mg/m{\ell}$ was added after penile corpus cavernosum were contracted by norepinephrine(NE) $1{\mu}M$. To analyze the mechanism of CF's vasorelaxation, CF extract infused into contracted penile tissues by NE after each treatment of indomethacin(IM), $N{\omega}$-nitro-L-arginine(L-NNA), methylene blue(MB), tetraethylammonium chloride(TEA).To study the effect of CF on influx of extracellular calcium chloride($Ca^{2+}$) in penile tissues, in $Ca^{2+}$-free krebs solution, $Ca^{2+}$ 1 mM infused into contracted penile tissues by NE after pretreatment of CF. Cytotoxic activity of CF on human umbilical vein endothelial cell(HUVEC) was measured by MTT assay, and nitric oxide(NO) prodution was measured by Griess reagent. CF relaxed cavernosal strip with endothelium contracted by NE, but in the strips without endothelium, CF-induced relaxation was significantly inhibited. The pretreatment of L-NNA, MB, TEA decreased significantly on the cavernosal relaxation than not-treatment of them. But the pretreatment of IM had no significant effect on the cavernosal relaxation. In $Ca^{2+}$-free krebs solution, when $Ca^{2+}$ infused into contracted penile tissues by NE, pretreatment of CF inhibit contraction induced by adding $Ca^{2+}$.NO production wasn't increased by treatment of CF on HUVEC. This findings showed that CF is effective for the relaxation of rabbit penile corpus cavernosum, and we suggest that CF relax rabbit corpus cavernosal smooth muscle through multiple action mechanisms that include increasing the release of nitric oxide from corporal sinusoidal endothelium, inhibition of $Ca^{2+}$ mobilization into cytosol from the extracellular fluid, and maybe a hyperpolarizing action.

• The Korean Journal of Physiology and Pharmacology
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• v.3 no.1
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• pp.83-91
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• 1999

Angiotensin II (ANG II) has a biphasic effect on $Na^+$ transport in proximal tubule: low doses of ANG II increase the $Na^+$ transport, whereas high doses of ANG II inhibit it. However, the mechanisms of high dose ANG II-induced inhibition on $Na^+$ uptake are poorly understood. Thus the aim of the present study was to investigate signal transduction pathways involved in the ANG II-induced inhibition of $Na^+$ uptake in the primary cultured rabbit renal proximal tubule cells (PTCs) in hormonally defined serum-free medium. ANG II $(10^{-9}\;M)-induced$ inhibition of $Na^+$ uptake was blocked by losartan $(10^{-8}\;M,\;AT_1\;antagonist),$ but not by PD123319 $(10^{-8}\;M,\;AT_2\;antagonist)$ (P<0.05). ANG II-induced inhibition of $Na^+$ uptake was also completely abolished by neomycin $(10^{-4}\;M,$ PLC inhibitor), W-7 $(10^{-4}\;M,$ calmodulin antagonist), and $AACOCF_3\;(10^{-6}\;M,\;PLA_2\;inhibitor)$ (P<0.05). ANG II significantly increased $[^3H]arachidonic$ acid (AA) release compared to control. The ANG II-induced $[^3H]AA$ release was blocked by losartan, $AACOCF_3,$ neomycin, and W-7, but not by PD123319. ANG II-induced $[^3H]AA$ release in the presence of extracellular $Ca^{2+}$ was greater than in $Ca^{2+}-free$ medium, and it was partially blocked by TMB-8 $(10^{-4}\;M,$ intracelluar $Ca^{2+}$ mobilization blocker). However, in the absence of extracellular $Ca^{2+},$ it was completely blocked by TMB-8. In addition, econazole $(10^{-6}\;M,$ cytochrome P-450 monooxygenase inhibitor) and indomethacin $(10^{-6}\;M,$ cyclooxygenase inhibitor) blocked ANG II-induced inhibition of $Na^+$ uptake, but NGDA $(10^{-6}\;M,$ lipoxygenase inhibitor) did not affect it. In conclusion, $PLA_2-mediated$ AA release is involved in ANG II-induced inhibition of $Na^+$ uptake and is modulated by $[Ca^{2+}]_i$ in the PTCs.

• Animal cells and systems
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• v.1 no.1
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• pp.81-86
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• 1997

We studied the mechanism of arachidonic acid on the secretion of a neurotransmitter in rat pheochromocytoma PC12 cells. Arachidonic acid inhibited the 70 mM $K^+$-induced secretion of norepinephrine. Arachidonic acid also inhibited the 70 mM $K^+$-induced $Ca^{2+}$ mobilization which is due to the opening of the voltage-sensitive $Ca^{2+}$ channels (VSCC). Both the half maximal inhibitory concentration ($IC_{50}$) of the norepinephrine secretion and VSCC coincided at 30 uM. The major oxidized metabolites of arachidonic acid, prostaglandins did not mimic the inhibitory effect of arachidonic acid. Nordihydroguaiaretic acid (NDGA) and indomethacin which are inhibitors of lipoxygenase and cyclooxygenase, respectively, did not block the inhibitory effect of arachidonic acid. The results suggest that arachidonic acid serves as a signal itself, not in the form of metabolites. The pretreatment of various $K^+$ channel blockers such as 4-aminopyridine, tetraethylarnmonium, glipizide, or glibenclamide also did not show any effect on the inhibitory effect of arachidonic acid. Through these results we suggest that arachidonic acid regulates VSCC directly and affects the secretion of neurotransmitters.

• Journal of Ginseng Research
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• v.37 no.2
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• pp.176-186
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• 2013

In this study, we have investigated the effects of total saponin from Korean red ginseng (TSKRG) on thrombin-induced platelet aggregation. TSKRG dose-dependently inhibited thrombin-induced platelet aggregation with $IC_{50}$ value of about 81.1 ${\mu}g/mL$. In addition, TSKRG dose-dependently decreased thrombin-elevated the level of cytosolic-free $Ca^{2+}$ ($[Ca^{2+}]_i$), one of aggregation-inducing molecules. Of two $Ca^{2+}$-antagonistic cyclic nucleotides as aggregation-inhibiting molecules, cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), TSKRG significantly dose-dependently elevated intracellular level of cAMP, but not cGMP. In addition, TSKRG dose-dependently inhibited thrombin-elevated adenosine triphosphate (ATP) release from platelets. These results suggest that the suppression of $[Ca^{2+}]_i$ elevation, and of ATP release by TSKRG are associated with upregulation of cAMP. TSKRG elevated the phosphorylation of vasodilator-stimulated phosphoprotein (VASP)-$Ser^{157}$, a cAMP-dependent protein kinase (A-kinase) substrate, but not the phosphorylation of VASP-$Ser^{239}$, a cGMP-dependent protein kinase substrate, in thrombin-activated platelets. We demonstrate that TSKRG involves in increase of cAMP level and subsequent elevation of VASP-$Ser^{157}$ phosphorylation through A-kinase activation to inhibit $[Ca^{2+}]_i$ mobilization and ATP release in thrombin-induced platelet aggregation. These results strongly indicate that TSKRG is a beneficial herbal substance elevating cAMP level in thrombin-platelet interaction, which may result in preventing of platelet aggregation-mediated thrombotic diseases.

• Journal of Ginseng Research
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• v.35 no.2
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• pp.209-218
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• 2011

Ginseng has been used as a general tonic agent to invigorate the human body as an adaptogenic agent. In a previous report, we have shown that ginseng contains a novel glycolipoprotein called gintonin. The main function of gintonin is to transiently enhance intracellular free $Ca^{2+}$ $[Ca^{2+}]_i$ levels in animal cells. The previous method for gintonin isolation included multiple steps using organic solvents. In the present report, we developed a simple method for the preparation of crude gintonin from ginseng root as well as stem and leaf, which produced a higher yield of gintonin than the previous one. The yield of gintonin was 0.20%, 0.29%, and 0.81% from ginseng root, stem, and leaf, respectively. The apparent molecular weight of gintonin isolated from stem and leaf through sodium dodecyl sulfate polyacrylamide gel electrophoresis was almost same as that from root but the compositions of amino acids, carbohydrates or lipids differed slightly between them. We also examined the effects of crude gintonin from ginseng root, stem, and leaf on endogenous $Ca^{2+}$-activated $Cl^-$ channel (CaCC) activity of Xenopus oocytes through mobilization of $[Ca^{2+}]_i$. We found that the order of potency for the activation of CaCC was ginseng root > stem > leaf. The $ED_{50}$ was $1.4{\pm}1.4$, $4.5{\pm}5.9$, and $3.9{\pm}1.1$ mg/mL for root, stem and leaf, respectively. In the present study, we demonstrated for the first time that in addition to ginseng root, ginseng stem and leaf also contain gintonin. Gintonin can be prepared from a simple method with higher yield of gintonin from ginseng root, stem, and leaf. Finally, these results demonstrate the possibility that ginseng stem and leaf could also be utilized for ginstonin preparation after a simple procedure, rather than being discarded.