• 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.

• Biomolecules & Therapeutics
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• v.24 no.5
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• pp.529-535
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• 2016

Atopic dermatitis (AD) results from gene and environment interactions that lead to a range of immunological abnormalities and breakdown of the skin barrier. Protease-activated receptor 2 (PAR2) belongs to a family of G-protein coupled receptors and is expressed in suprabasal layers of the epidermis. PAR2 is activated by both trypsin and a specific agonist peptide, SLIGKV-$NH_2$ and is involved in both epidermal permeability barrier homeostasis and epithelial inflammation. In this study, we investigated the effect of lobaric acid on inflammation, keratinocyte differentiation, and recovery of the skin barrier in hairless mice. Lobaric acid blocked trypsin-induced and SLIGKV-$NH_2$-induced PAR2 activation resulting in decreased mobilization of intracellular $Ca^{2+}$ in HaCaT keratinocytes. Lobaric acid reduced expression of interleukin-8 induced by SLIGKV-$NH_2$ and thymus and activation regulated chemokine (TARC) induced by tumor necrosis factor-a (TNF-${\alpha}$) and IFN-${\gamma}$ in HaCaT keratinocytes. Lobaric acid also blocked SLIGKV-$NH_2$-induced activation of ERK, which is a downstream signal of PAR2 in normal human keratinocytes (NHEKs). Treatment with SLIGKV-$NH_2$ downregulated expression of involucrin, a differentiation marker protein in HaCaT keratinocytes, and upregulated expression of involucrin, transglutamase1 and filaggrin in NHEKs. However, lobaric acid antagonized the effect of SLIGKV-$NH_2$ in HaCaT keratinocytes and NHEKs. Topical application of lobaric acid accelerated barrier recovery kinetics in a SKH-1 hairless mouse model. These results suggested that lobaric acid is a PAR2 antagonist and could be a possible therapeutic agent for atopic dermatitis.

• The Korean Journal of Physiology and Pharmacology
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• v.15 no.3
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• pp.129-135
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• 2011

In this study we determined whether or not 5-hydroxytryptamine (5-HT) has an effect on the pacemaker activities of interstitial cells of Cajal (ICC) from the mouse small intestine. The actions of 5-HT on pacemaker activities were investigated using a whole-cell patch-clamp technique, intracellular $Ca^{2+}$ ($[Ca^{2+}]_i$) analysis, and RT-PCR in ICC. Exogenously-treated 5-HT showed tonic inward currents on pacemaker currents in ICC under the voltage-clamp mode in a dose-dependent manner. Based on RT-PCR results, we found the existence of 5-$HT_{2B,\;3,\;4,\;and\;7}$ receptors in ICC. However, SDZ 205557 (a 5-$HT_4$ receptor antagonist), SB 269970 (a 5-$HT_7$ receptor antagonist), 3-tropanylindole - 3 - carboxylate methiodide (3-TCM; a 5-$HT_3$ antagonist) blocked the 5-HT-induced action on pacemaker activity, but not SB 204741 (a 5-$HT_{2B}$ receptor antagonist). Based on $[Ca^{2+}]_i$ analysis, we found that 5-HT increased the intensity of $[Ca^{2+}]_i$. The treatment of PD 98059 or JNK II inhibitor blocked the 5-HT-induced action on pacemaker activity of ICC, but not SB 203580. In summary, these results suggest that 5-HT can modulate pacemaker activity through 5-$HT_{3,\;4,\;and\;7}$ receptors via $[Ca^{2+}]_i$ mobilization and regulation of mitogen-activated protein kinases.

• Journal of Applied Biological Chemistry
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• v.66
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• pp.402-407
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• 2023

The regulation of platelet aggregation is crucial for maintaining normal hemostasis, but abnormal or excessive platelet aggregation can contribute to cardiovascular disorders such as stroke, atherosclerosis, and thrombosis. Therefore, identifying substances that can control or suppress platelet aggregation is a promising approach for the prevention and treatment of these conditions. Artemisinin, a compound derived from Artemisia or Scopolia plants, has shown potential in various areas such as anticancer and Alzheimer's disease research. However, the specific role and mechanisms by which artemisinin influences platelet activation and thrombus formation are not yet fully understood. This study investigated the effects of artemisinin on platelet activation and thrombus formation. As a result, cAMP production were increased significantly by artemisinin, as well as phosphorylated VASP and IP₃R which are substrates to cAMP-dependent kinase by artemisinin in a significant manner. The Ca²⁺ normally mobilized from the dense tubular system was inhibited due to IP₃R phosphorylation from artemisinin, and phosphorylated VASP by artemisinin aided in inhibiting platelet activity via αIIb/β3 platelet membrane inactivation and inhibiting fibrinogen binding. Finally, artemisinin inhibited thrombin-induced thrombus formation. Therefore, we suggest that artemisinin has importance with cardiovascular diseases stemming from the abnormal platelets activation and thrombus formation by acting as an effective prophylactic and therapeutic agent.

• Journal of Ginseng Research
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• v.41 no.4
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• pp.548-555
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• 2017

Background: Korean Red Ginseng has been used for several decades to treat many diseases, enhancing both immunity and physical strength. Previous studies have documented the therapeutic effects of ginseng, including its anticancer, antiaging, and anti-inflammatory activities. These activities are mediated by ginsenosides present in the ginseng plant. Ginsenoside Rg3, an effective compound from red ginseng, has been shown to have antiplatelet activity in addition to its anticancer and anti-inflammatory activities. Platelets are important for both primary hemostasis and the repair of the vessels after injury; however, they also play a crucial role in the development of acute coronary diseases. We prepared ginsenoside Rg3-enriched red ginseng extract (Rg3-RGE) to examine its role in platelet physiology. Methods: To examine the effect of Rg3-RGE on platelet activation in vitro, platelet aggregation, granule secretion, intracellular calcium ($[Ca^{2+}]_i$) mobilization, flow cytometry, and immunoblot analysis were carried out using rat platelets. To examine the effect of Rg3-RGE on platelet activation in vivo, a collagen plus epinephrine-induced acute pulmonary thromboembolism mouse model was used. Results: We found that Rg3-RGE significantly inhibited collagen-induced platelet aggregation and $[Ca^{2+}]_i$ mobilization in a dose-dependent manner in addition to reducing ATP release from collagen-stimulated platelets. Furthermore, using immunoblot analysis, we found that Rg3-RGE markedly suppressed mitogen-activated protein kinase phosphorylation (i.e., extracellular stimuli-responsive kinase, Jun N-terminal kinase, p38) as well as the PI3K (phosphatidylinositol 3 kinase)/Akt pathway. Moreover, Rg3-RGE effectively reduced collagen plus epinephrine-induced mortality in mice. Conclusion: These data suggest that ginsenoside Rg3-RGE could be potentially be used as an antiplatelet therapeutic agent against platelet-mediated cardiovascular disorders.

• Biomolecules & Therapeutics
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• v.20 no.5
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• pp.463-469
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• 2012

Atopic dermatitis is a chronic, inflammatory disease of the skin with increased transepidermal water loss. Both an abnormal inflammatory response and a defective skin barrier are known to be involved in the pathogenesis of atopic dermatitis. Protease activated receptor 2 (PAR2) belongs to a family of G-protein coupled receptors and is activated by both trypsin and a specific agonist peptide, SLIGKV-$NH_2$. PAR2 is expressed in suprabasal layers of the epidermis and regulates inflammatory responses and barrier homeostasis. In this study, we show that nordihydroguaiaretic acid (NDGA) inhibits the PAR2-mediated signal pathway and plays a role in skin barrier recovery in atopic dermatitis. Specifically, NDGA reduces the mobilization of intracellular $Ca^{2+}$ in HaCaT keratinocytes by down-regulating inflammatory mediators, such as interleukin-8, thymus and activation-regulated chemokine and intercellular cell adhesion molecule-1 in HaCaT keratinocytes. Also, NDGA decreases the protein expression of involucrin, a differentiation maker of keratinocyte, in both HaCaT keratinocytes and normal human epidermal keratinocytes. We examined NDGA-recovered skin barrier in atopic dermatitis by using an oxazolone-induced atopic dermatitis model in hairless mice. Topical application of NDGA produced an increase in transepidermal water loss recovery and a decrease in serum IgE level, without weight loss. Accordingly, we suggest that NDGA acts as a PAR2 antagonist and may be a possible therapeutic agent for atopic dermatitis.

• Journal of Microbiology and Biotechnology
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• v.7 no.4
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• pp.229-236
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• 1997

A gene, $phbC_{2.4.1}$ encoding poly-3-hydroxybutyric acid (PHB) synthase of Rhodobacter sphaeroides 2.4.1 was cloned by employing heterologous expression in Escherichia coli. R. sphaeroides chromosomal DNA partially digested with MboI was cloned in pUC19 followed by mobilization into E. coli harbouring $phbA,B_{AC}$ in pRK415, which code for ${\beta}$-ketothiolase and acetoacetyl CoA reductase of Alcaligenes eutrophus, respectively. Two E. coli clones carrying R. sphaeroides chromosomal fragment of $phbC_{2.4.1}$ in pUC19 were selected from ca. 10,000 colonies. The PHB-producing colonies had an opaque white appearance due to the intracellular accumulation of PHB. The structure of PHB produced by the recombinant E. coli as well as from R. sphaeroides 2.4.1 was confirmed by [$H^{+}$]-nuclear magnetic resonance (NMR) spectroscopy. Restriction analysis of the two pUC19 clones revealed that one insert DNA fragment is contained as a part of the other cloned fragment. An open reading frame of 601 amino acids of $phbC_{2.4.1}$ with approximate M.W. of 66 kDa was found from nucleotide sequence determination of the 2.8-kb SaiI-PstI restriction endonuclease fragment which had been narrowed down to support PHB synthesis through heterologous expression in the E. coli harbouring $phbA,B_{AC}$. The promoter (s) of the $phbC_{2.4.1}$ were localized within a 340-bp DNA region upstream of the $phbC_{2.4.1}$ start codon according to heterologous expression analysis.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1996.11a
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• pp.75-84
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• 1996

Platelets from a patient with a mild inherited bleeding disorder and abnormal platelet aggregation and secretion show reduced generation of inositol 1,4,5-trisphosphate (IP$_3$), mobilization of intracellular Ca$\^$2+/, and phosphorylation of pleckstrin in response to several G protein mediated agonists, suggesting a possible defect at the level of phospholipase C (PLC) activation. A procedure was developed that allows quantitation of platelet PLC isozymes. After fractionation of platelet extracts by high-performance liquid chromatography, seven, out often known PLC isoforms were detected by immunoblot analysis. The amount of these isoforms in normal platelets decreased in the order PLC-${\gamma}$2 > PLC-${\beta}$2 > PLC-${\beta}$3 > PLC-${\beta}$l > PLC-${\gamma}$ > PLC-$\delta$1 > PLC-${\beta}$4. Compared with normal platelets, platelets from the patient contained approximately one-third the amount of PLC-${\beta}$2, whereas PLC-${\beta}$4 was increased threefold. These results suggest that the impaired platelet function in the patient in response to multiple G protein mediated agonists is attributable to a deficiency of PLC-${\beta}$2. They document for the first time a specific PLC isozyme deficiency in human platelets and provide an unique opportunity to understand the role of different PLC isozymes in normal platelet function.

• Journal of Ginseng Research
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• v.45 no.4
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• pp.490-497
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• 2021

Background and objective: Synthetic ginsenoside compounds G-Rp (1,3, and 4) and natural ginsenosides in Panax ginseng 20(S)-Rg3, Rg6, F4 and Ro have inhibitory actions on human platelets. However, the inhibitory mechanism of ginsenoside Rk1 (G-Rk1) is still unclear thus, we initiated investigation of the anti-platelet mechanism by G-Rk1 from Panax ginseng. Methodology: Our study focused to investigate the action of G-Rk1 on agonist-stimulated human platelet aggregation, inhibition of platelet signaling molecules such as fibrinogen binding with integrin α_IIbβ₃ using flow cytometry, intracellular calcium mobilization, fibronectin adhesion, dense granule secretion, and thromboxane B2 secretion. Thrombin-induced clot retraction was also observed in human platelets. Key Results: Collagen, thrombin, and U46619-stimulated human platelet aggregation were dose-dependently inhibited by G-Rk1, while it demonstrated a more effective suppression on collagen-stimulated platelet aggregation using human platelets. Moreover, G-Rk1 suppressed collagen-induced elevation of Ca²⁺ release from endoplasmic reticulum, granule release, and α_IIbβ₃ activity without any cytotoxicity. Conclusions and implications: These results indicate that G-Rk1 possess strong anti-platelet effect, proposing a new drug candidate for treatment and prevention of platelet-mediated thrombosis in cardiovascular disease.