• Journal of Physiology & Pathology in Korean Medicine
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• v.27 no.6
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• pp.759-763
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• 2013

This experiment was performed to investigate whether 50% ethanol extracts of the combined-preparation of Longanae Arilus, Chrysanthemi Flos, Zizyphi Fructus and Ginseng Radix alba (CPE) has hypnotic effects and/or enhances pentobarbital-induced sleeping time. Locomotor activity was evaluated using a ambulometer of tilting-type. The sedative-hypnotic effects were evaluated by measuring the sleeping onset time and sleeping time in pentobarbital-treated mice 30 min. after oral administration of CPE and muscimol. The intracellular $Cl^-$ concentration of cerebellar granule cells was estimated using $Cl^-$ sensitive fluorescence probe N-(ethoxycarbonylmethyl)-6-methoxyquinolinium (MQAE). CPE (150 mg/kg) decreased the locomotor activity, but CPE itself did not induce sleep. However, CPE reduced sleeping onset and prolonged sleeping time induced by pentobarbital (42 mg/kg). In addition, CPE (2 ${\mu}g/ml$) and pentobarbital (2.5 ${\mu}M$) itself did not affect on the chloride influx in primary cultured cerebellar granule cells, but the combination of CPE and pentobarbital (2.5 ${\mu}M$) increased the chloride influx onto the cells. In conclusion, it is suggested that CPE might augment pentobarbital-induced sleep through the increase of chloride influx.

• Animal cells and systems
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• v.7 no.1
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• pp.75-79
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• 2003

Highly purified high performance thin layer chromatography (HPTLC) fractions containing a putative calcium influx factor (CIF) were prepared from the Jurkat cells and Xenopus oocytes in which $Ca^{2+}$ stores were depleted by thapsigargin treatment and from the yeast in which intracellular $Ca^{2+}$ stores were also depleted by genetic means. Microinjection of the fractions has been shown to elicit $Ca^{2+}$ dependent currents in Xenopus oocytes. The nature of the membrane currents evoked by the putative CIF appeared to be carried by chloride ions since the current was blocked by the selective chloride channel blocker 1 mM niflumic acid and its reversal potential was about -24 mV. Injection of the calcium chelator 1,2-bis(2-aminophenoxy)ethane-N, N, N',N'-tetraacetic acid (BAPTA) eradicated the current activities, suggesting the current responses are entirely $Ca^2$-dependent. Moreover, the currents were sensitive to the removal of extracellular calcium, indicating the dependence on calcium entry through the plasma membrane calcium entry channels. CIF activities were insensitive to protease, heat, and acid treatments and to Dische-reaction whereas the activities were sensitive to nucleotide pyrophosphatase and hydrazynolysis. The fraction might have a sugar because it was sensitive to Molisch test and Seliwaniff's resorcinol reaction. From the above results, CIF as a small and stable molecule seems to have pyrimidine, pyrophosphate, and a sugar moiety.oiety.

• The Korean Journal of Pharmacology
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• v.24 no.1
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• pp.71-81
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• 1988

Glutamate and aspartate may evoke an increase in membrane permeability to monovalent cations and $Ca^{++}$. However, it is uncertain whether $Ca^{++}$ influx is mediated by voltage dependent $Ca^{++}$ channels or by excitatory amino acid activated channels. In addition, the influences of excitatory amino acids on $Ca^{++}$ uptake by neuronal tissues as well as the responses of their actions to extracellular $Mg^{++}$ concentration are different. $K^{+}$ induced $Ca^{++}$ uptake by synaptosomes was dependent on extracellular $Mg^{++}$ up to 5 mM and at concentration of 10 mM, $Ca^{++}$ influx was rather reduced. In $Na^{+}$ rich media, glutamate-and aspartate-induced $Ca^{++}$ uptake was increased by $Mg^{++}$ in a dose independent manner. However, the response for NMDA was inhibited by $Mg^{++}$ at concentrations above 2 mM. $K^+$-and glutamate-induced $Ca^{++}$ influx s were inhibited by 2,4-dinitrophenol, chlorprom-azine and verapamil but not by tetraethylammonium chloride. Tetrodotoxin effectively inhibited the action of glutamate but did not affect that of $K^+$. The response for MNDA was inhibited by 2, 4-dinitrophenol and tetrodotoxin, slightly inhibited by verapamil, and not affected by tetraethylammonium chloride. In $Na^{++}$ rich medium, depolarizing action of glutamate, aspartate and MNDA on synaptosomes was not demonstrated, whereas these agents stimulated $Ca^{++}$ uptake and caused $Ca^{++}$ influx induced depolarization at mitochondria. On the other hand, the activities of synaptosomal ATPases were not affected by excitatory amino acids at 5 mM. The results suggest that glutamate or NMDA induced $Ca^{++}$ influx at synaptosomes exhibits different responses for extracellular $Mg^{++}$ Ex citatory amino acids induced $Ca^{++}$ influx at synaptosomes may be associated with increased permeability of membrane for $Na^{++}$ and $Ca^{++}$ except $K^{++}$ and membrane depolarization due to increased ionic permeability.

• Journal of Physiology & Pathology in Korean Medicine
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• v.25 no.6
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• pp.996-999
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• 2011

This experiment was performed to investigate whether the combined-preparation of crude drugs (The $Well^{TM}$ Preparation, TW), has hypnotic effects and/or enhances pentobarbital-induced sleep behaviors. TW was mixed with water extracts of Ginseng Radix red, Germinated brown rice, cultured mountain ginseng, and 50% ethanol extracts of Longanae Arillus, Nelumbinis Folium and Chrysanthemi Flos. TW (100 mg/kg, p.o.) reduced sleep onset and prolonged sleep time induced by pentobarbital similar to muscimol (0.2 ${\mu}M$), a $GABA_A$ receptor agonist. Also, TW (2 ${\mu}g$/ml) and pentobarbital (2.5 ${\mu}M$) did not affect the chloride influx in primary cultured cerebellar granule cells, respectively, but the combined-treatment of TW (2 ${\mu}g$/ml) and pentobarbital (2.5 ${\mu}M$) increased the chloride influx onto the cells. In conclusion, TW augments pentobarbital-induced sleep behaviors; these effects may result from chloride channel activation.

• Biomolecules & Therapeutics
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• v.16 no.3
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• pp.261-269
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• 2008

This experiment was performed to investigate the anxiolytic-like effects of cyclopeptide fraction alkaloids of Zizyphi Spinosi Semen (CFAZ), by using the experimental paradigms of anxiety, and compared with those of a known anxiolytic, diazepam. CFAZ (8.0 mg/kg, p.o.) increased the percentage of time spent on the open arms and the number of open arms entries in the elevated plus-maze test, increased the number of head dips in the hole-board test, and increased the percentage of center zone ambulatory time in the open-field box. However, CFAZ has no effect on the locomotor activity, while diazepam (2.0 mg/kg, p.o.) significantly reduced locomotor activity. CFAZ did not influence the grip force in the grip strength meter test, either. From the molecular experiments, CFAZ increased chloride influx in cultured cerebellar granule cells. In addition, $GABA_A$ receptors $\gamma$-subunit were over-expressed by CFAZ in cultured cerebellar granule cells. It is concluded that CFAZ may have anxiolytic-like effects, and these effects may be mediated by $GABA_A$ receptors.

• Natural Product Sciences
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• v.18 no.2
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• pp.67-75
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• 2012

Zizyphi Spinosi Semen (ZSS) have been widely used for the treatment of insomnia in Asia. This experiment was performed to investigate whether methanol extract of ZSS (MEZSS) has hypnotic effects through the ${\gamma}$-amino butyric acid (GABA)ergic systems. MEZSS inhibited the locomotor activity. MEZSS enhanced pentobarbital-induced sleep behaviors. However, MEZSS itself did not induce sleep at higher dose, similar to muscimol. On the other hand, both pentobarbital and MEZSS increased the non rapid eye move (NREM) sleep, especially reducing the -wave electroencephalogram (EEG) activity in REM sleep. MEZSS showed similar effects with muscimol on potentiating chloride influx induced by pentobarbital. MEZSS significantly increased GABAA receptors ${\gamma}$-subunit expression and slightly decreased ${\beta}$-subunit expression in hypothalamus and thalamus, showing that subunit-expression was similar to diazepam. In addition, MEZSS enhanced the expression of glutamic acid decarboxylase (GAD). In conclusion, it is suggested that MEZSS might augment pentobarbital-induced sleep behaviors through the modification of GABAergic systems.

• Natural Product Sciences
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• v.19 no.2
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• pp.179-185
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• 2013

Polygalae radix (PR) has traditionally been used as a sedative and anti-stress agent in oriental countries for a long time. PR which contains many ingredients is especially rich in saponins. This study was performed to investigate whether ethanol extract of PR enhances pentobarbital-induced sleep behaviors. In addition, possible mechanisms also were investigated. PR inhibited locomotor activity in mice. PR increased sleep rate and sleep time by concomitant administration with sub-hypnotic dose of pentobarbital (28 mg/kg). PR prolonged total sleeping time, and shortened sleep latency induced by pentobarbital (42 mg/kg). In addition, PR increased intracellular chloride concentration in primary cultured neuronal cells. The expression level of glutamic acid decarboxylase (GAD) were increased, and ${\gamma}$-aminobutyric acid $(GABA)_A$ receptors subunits were modulated by PR, especially increasing ${\gamma}$-subunit expression. In conclusion, PR augments penobarbital-induced sleep behaviors through activation of $GABA_A$ receptors and chloride channel complex.

• Journal of Physiology & Pathology in Korean Medicine
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• v.27 no.4
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• pp.437-445
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• 2013

This study was investigated to evaluate the vasorelaxant effect of Rubus coreanus(RC) extract on contracted rabbit carotid artery and its mechanism. To study the effect of RC extract on contracted rabbit carotid arterial strips, arterial strips with intact or damaged endothelium were used for experiment using organ bath. The pre-contracted arterial strips with norepinephrine(NE) or potassium chloride(KCl) was treated with various concentrations of an extract of RC(0.01, 0.03, 0.1, 0.3 and 1.0 $mg/m{\ell}$). To determine the mechanisms of RC-induced vasorelaxant, RC extract was infused into contracted arterial rings which had been pretreated by indomethacin(IM), tetraethylammonium chloride(TEA), $N{\omega}$-nitro-L-arginine (L-NNA), methylene blue(MB). And calcium chloride(Ca) 1 mM was infused into precontracted arterial ring induced by NE or KCl after treatment of RC extract in $Ca^{2+}$-free krebs solution. Cytotoxic activity of RC extract on human umbilical vein endothelial cell(HUVEC) was measured by MTT assay, and nitric oxide(NO) prodution was measured by Griess reagent. RC extract revealed significant relaxation on NE-induced arterial contraction, but didn't relax on KCl-induced arterial contraction. RC extract also had an effective relaxation to the intact endothelium arterial ring, but not the damaged endothelium arterial ring. Treatment of IM, TEA, L-NNA, MB reduced the relaxation of RC extract. Pretreatment of RC extract inhibited the contraction by influx of extracellular $Ca^{2+}$ in contracted arterial ring induced by NE, but it didn't work the contraction by influx of extracellular $Ca^{2+}$ in contracted arterial ring induced by KCl in $Ca^{2+}$-free krebs solution. RC extract increased nitric oxide production on HUVEC. This study indicated that the relaxation effect of RC extract on contracted rabbit carotid artery is related with NO-cGMP pathway, EDHF, prostacyclin.

• Biomolecules & Therapeutics
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• v.15 no.3
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• pp.175-181
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• 2007

Zizyphi Spinosi Semen (ZSS), a traditional Chinese folk medicine, has been used for treatment of insomnia and anxiety. This experiment was performed to investigate the anxiolytic-like effect of methanol extract of ZSS (MEZSS) in mice by using the experimental paradigms of anxiety and compared with that of a known anxiolytic, diazepam. In the elevated plus-maze test, it showed that MEZSS (100 mg/kg, p.o.) and diazepam (2.0 mg/kg, p.o.) increased the percentage of time spent on the open arms and the number of open arms entries. MEZSS (50, 100 and 200 mg/kg, p.o.) and diazepam (0.5 mg/kg, p.o.) significantly increased the number of head dips compared with that of control group in the hole-board test. However, MEZSS has no effect on decreasing the locomotor activity, while diazepam (2.0 mg/kg, p.o.) significantly inhibited locomotor activity. MEZSS did not decrease the strength force in the grip strength test, either. In addition, GABAergic involvements were also investigated to understand the possible mechanisms. $GABA_{A}$ receptors subunits and glutamic acid decarboxylase (GAD) were not over expressed, compared with that of the saline group. We also found that MEZSS did not increase chloride influx in cultured cerebellar granule cells. It is concluded that MEZSS might have anxiolytic-like effects, but these effects might not be mediated by GABAergic transmission.

• The Korea Journal of Herbology
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• v.30 no.6
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• pp.25-32
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• 2015

Objectives : The purpose of this study was to investigate the endothelium-dependent vasorelaxation effect of Cynomorii Herba(CH) extract on contracted rabbit carotid artery.Methods : To clarify the vasorelaxation effect of CH extract, arterial strips with intact was used, to endothelium -dependent vasorelaxation effect of CH extract, arterial strips damaged endothelium was used for experiment using organ bath. Arterial strips was contracted with phenylephrine(PE) before treated with CH extract(0.01, 0.03 and 0.1 ㎎/㎖). To study mechanisms of CH-induced vasorelaxation effect, CH extract infused into arterial rings after treatment by indomethacin(IM), tetraethylammonium chloride(TEA), Nω-nitro-L-arginine (L-NNA), methylene blue(MB) for comparing with non-treated. And calcium chloride(Ca²⁺) 1 mM was treated into precontracted arterial ring induced by PE after treatment of CH extract in Ca²⁺-free krebs solution. Cytotoxic activity of CH extract on human umbilical vein endothelial cell(HUVEC) was measured by MTT assay, and nitric oxide(NO) concentration was measured by Griess reagent.Results : PE-induced arterial strips was significantly relaxed, but the damaged endothelium arterial ring wasn't relaxed by CH extract. Pretreatment of IM, TEA didn't inhibit the vasorelaxation of CH extract, but pretreatment of L-NNA, MB inhibited the vasorelaxation of CH extract. Pretreatment of CH extract reduced the increase of contraction by influx of extracellular Ca²⁺ in contracted arterial ring induced by PE, CH extract increased nitric oxide concentration on HUVEC significantly.Conclusions : This study shows that CH extract have the vasorelaxation effect by blocking the influx of extracellular Ca²⁺ through the activating NO-cGMP system.