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Aim To examine the effect of oral diabetes medication on the risk of dementia in an elderly cohort with type 2 diabetes. Methods This was a population-based cohort study using the Korean National Health Insurance claims data from 2002 to 2013. Elderly subjects (60 years of age or older) with and without type 2 diabetes were included; patients with new-onset type 2 diabetes were further divided into the oral diabetes medication group and no-medication group. Results Among 278,290 patients with type 2 diabetes, 56,587 developed dementia (20.3%) over 11 years of follow-up. Type 2 diabetes was associated with a 1.69-fold increased risk of dementia (95% CI 1.66–1.72). Among patients with newly diagnosed type 2 diabetes, the risk of dementia was lower in the oral diabetes medication group than in the no-medication group (adjusted hazard ratio [aHR], 0.79; 95% CI 0.77–0.81). Lower risk of dementia was particularly noticeable in all of the combination therapy groups and especially lower in the combination therapy group treated with dipeptidyl peptidase 4 inhibitor (aHR 0.48, 95% CI 0.45–0.51). Conclusion Overall, the use of oral diabetes medication in type 2 diabetes patients significantly decreased the risk of dementia.

The D1 dopamine receptor agonist, SKF-38393, induces cytotoxicity in striatal dopaminergic neurons via an extracellular signal-regulated kinase (ERK) signaling cascade. However, the underlying mechanism remains unclear. We hypothesized that repeated activation of dopaminergic receptors by agonists could lead to neuronal cell death. This study investigated the effects of SKF-38393 on dopaminergic neuronal cell death in a 6-hydroxydopamine-lesioned rat model of Parkinson’s disease (PD) and PC12 cells. In the PD model, SKF-38393 administration (3 and 10 mg/kg per day, s.c.) for 8 weeks significantly increased the number of tyrosine hydroxylase-immunopositive neuronal cells in nigrostriatal regions. SKF-38393 administration for 8 weeks induced phosphorylation of sustained ERK1/2 and Bad (Bcl-2-associated death promoter) at Ser155 (BadSer155), and augmented Bax (Bcl-2-associated X protein) expression. However, SKF-38393 only increased Bad phosphorylation at Ser112 (BadSer112) when administered for 4 weeks. In PC12 cells, toxic levels of SKF-38393 (20 and 50 μM) rapidly induced formation of neurite-like processes, but not in the presence of an adenylyl cyclase inhibitor (MDL-12330 A). SKF-38393 (20 and 50 μM) induced sustained ERK1/2 and BadSer155 phosphorylation as well as caspase-3 activation. At a non-toxic level (5 μM), SKF-38393 produced only transient ERK1/2 and BadSer112 phosphorylation. Repeated treatments with SKF-38393 (5 μM) for 1–3 days activated BadSer112. Repeated treatments for 4–7 days induced sustained ERK1/2 and BadSer155 phosphorylation as well as Bax and caspase-3 activation. These results suggest that SKF-38393 induces neurotoxicity by activation of the sustained ERK–Bad–Bax system. These findings contribute to an understanding of the adverse effects of D1 dopamine receptor agonists in patients with PD.

Background The ethanol extract of Gynostemma pentaphyllum Makino leaves (EGP) has been reported recently to have anxiolytic effects on chronically stressed mice models. Purpose We aimed to investigate the efficacy and safety of EGP on anxiety level in healthy Korean subjects under chronic stressful conditions. Study design Double-blind, placebo-controlled trial. Methods This study was conducted with 72 healthy adults who had perceived chronic stress and anxiety with a score on the State-Trait Anxiety Inventory (STAI) from 40 to 60. Participants were randomly assigned to receive either EGP (200 mg, twice a day, N = 36) or placebo (N = 36). All participants were exposed to repetitive loads of stress by performing the serial subtraction task for 5 min every second day during the 8-week intervention. Primary outcome of Trait-STAI and secondary outcomes of State-STAI, total score of STAI, Hamilton Anxiety Inventory (HAM-A), Beck Anxiety Inventory (BAI), blood norepinephrine and adrenocorticotropic hormone (ACTH), salivary cortisol and alpha-amylase, cardiovascular autonomic nervous system (ANS) functional test, and heart rate variability (HRV) test were measured before and after intervention. Results After the 8-week intervention, the EGP significantly lowered the score of the Trait Anxiety Scale of the STAI (T-STAI) by 16.8% compared to the placebo (p = 0.041). The total score on the STAI decreased by 17.8% in the EGP group and tended to improve compared with that of the placebo group (p = 0.067). There were no significant differences in the changes in score of S-STAI, HAM-A, BAI, and other parameters from baseline between the two groups. There was no causal relationship between the ingestion of EGP and adverse drug reactions. Conclusion We found that supplementation with EGP reduced “anxiety proneness” in subjects under chronic psychological stress, as shown by a decrease in the score of T-STAI and the tendency for decrease in the total score of STAI. This result suggests that EGP supplementation can be used as a regimen to safely reduce stress and anxiety; however, more studies are needed to establish the long-term safety and effectiveness.

Aims Asian patients are known to be more prone to bleeding complications than patients of other ethnicities. Therefore, there are possibilities of other risk factors that should be given special consideration for dosage adjustment in this specific ethnic group. This study aimed to investigate the risk factors for bleeding complications in Asian patients under appropriate edoxaban dosage regimens. Methods Data on patients taking proper dosages, based on the Lixiana package insert, were analysed. Univariate and multivariable analyses were conducted to evaluate associations between risk factors and bleeding outcomes. Subgroup analysis was performed on high-risk patients for bleeding complications whose edoxaban dose was reduced according to the package insert. Results In total, 346 patients were included. Among them, 32 patients experienced bleeding complications. Patients with weight ≤60 kg and with cancer showed around 3.3- and 3.4-fold increased risk of bleeding complications compared to heavier patients (>60 kg) and those without cancer, respectively. In subgroup analysis with high-risk patients who took low-dose edoxaban (15 and 30 mg), weight ≤60 kg remained a significant factor for bleeding outcomes. Conclusion This study showed that weight ≤60 kg and the presence of cancers could affect bleeding complications, which occurred despite proper edoxaban treatment in Asian patients. Therefore, more strict dosage guideline could be considered in populations with high proportions of Asian ethnicities.

Gynosaponins have pharmacological effects on 3,4-L-dihydroxyphenylalanine (L-DOPA)-related or dopamine-related neurological diseases; however, the neuroprotective functions of single compound of gynosaponins remain undefined. This study investigated the cytotoxic effects of gynosaponin TN-2 on L-DOPA in pheochromocytoma 12 cells. Gynosaponin TN-2, at 0.5-3 μM, did not exhibit cytotoxicity and protected against L-DOPA (100 and 200 μM)-induced cell death. Gynosaponin TN-2 (0.5 and 1.0 μM) inhibited the L-DOPA (100 and 200 μM)-induced sustained extracellular signal-regulated protein kinases 1 and 2 phosphorylation. Gynosaponin TN-2 at 0.5 and 1.0 μM also reduced L-DOPA (100 and 200 μM)-induced JNK1/2 phosphorylation and cleaved caspase-3 expression. These results suggested that gynosaponin TN-2 exerts protective effects on L-DOPA (100 and 200 μM)-induced apoptotic cell death by modulating extracellular signal-regulated protein kinases 1 and 2 activation in pheochromocytoma 12 cells.

Purpose: Although moderate- to high-intensity statin therapy is increasingly recommended in cardiovascular disease patients, the efficacy and safety in elderly patients have not been proven clearly. Here, we compare the effect of various-intensity statins between elderly and very elderly patients. Methods: 43,870 patients over 65 years old who were treated with statins were screened using electronic medical record data. Results: We evaluated 451 patients in the elderly group aged 65–74 years and 159 patients in the very elderly group over 75 years old. Baseline cholesterol profiles were similar between the 2 groups, but the 10-year atherosclerotic cardiovascular disease (ASCVD) risk was significantly higher in the very elderly (20.9 ± 11.5% vs. 37.2 ± 13.6%, p < 0.001). The reduction rate of low-density lipoprotein (LDL) (–40.2 ± 21.3% vs. –39.3 ± 21.0%, p = 0.634) and the ratio of target LDL attainment (74.2 vs. 79.2%, p = 0.252) were similar between the 2 groups. Low-intensity statins showed comparable LDL cholesterol reduction with moderate-intensity statins both in the elderly and the very elderly groups. The 10-year ASCVD risk reduction was similar between the 2 groups (–3.5 ± 4.9% vs. –3.0 ± 8.4%, p = 0.480), but in the very elderly group, no different ASCVD reduction rate was shown in low- to high-intensity statins (p = 0.784). Only the elderly group showed a significant correlation (r = 0.112, p = 0.017) with LDL reduction and 10-year ASCVD risk. Interestingly, the incidence of adverse drug reaction (ADR) was higher in the very elderly group (4.4%) than in the elderly group (2.7%) and was more frequent in high-intensity statin therapy. Conclusion: The efficacy of statins in LDL reduction was similar between the elderly and very elderly population. However, the benefit of moderate- to high-intensity statins is limited considering potential ADR. Therefore, the stepwise intensification of statin therapy might be necessary for the very elderly in spite of the higher cardiovascular risk.

Background Previous studies have revealed that gypenosides (GPS) improve the symptoms of anxiety disorders in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned rat model of Parkinson’s disease (PD). The present study aimed to investigate the effects of GPS on memory deficits in an MPTP-lesioned mouse model of PD treated with L-3,4-dihydroxyphenylalanine (L-DOPA). Methods MPTP (30 mg/kg/day, 5 days)-lesioned mice were treated with GPS (50 mg/kg) and/or L-DOPA (10 and 25 mg/kg) for 21 days. After the final treatments, behavioral changes were assessed in all mice using passive avoidance and elevated plus-maze tests. We then evaluated the biochemical influences of GPS treatment on levels of tyrosine hydroxylase (TH), dopamine, N-methyl-D-aspartate (NMDA) receptors, extracellular signal-regulated kinase (ERK1/2), and cyclic AMP-response element binding protein (CREB) phosphorylation. Results MPTP-lesioned mice exhibited deficits associated with habit learning and spatial memory, which were further aggravated by treatment with L-DOPA (25 mg/kg). However, treatment with GPS (50 mg/kg) ameliorated memory deficits. Treatment with GPS (50 mg/kg) also improved L-DOPA (25 mg/kg)-treated MPTP lesion-induced decreases in retention latency on the passive avoidance test, as well as levels of TH-immunopositive cells and dopamine in the substantia nigra and striatum. GPS treatment also attenuated increases in retention transfer latency on the elevated plus-maze test and in NMDA receptor expression, as well as decreases in the phosphorylation of ERK1/2 and CREB in the hippocampus. Treatment with L-DOPA (10 mg/kg) also ameliorated deficits in habit learning and spatial memory in MPTP-lesioned mice, and this effect was further enhanced by treatment with GPS (50 mg/kg). Conclusion GPS ameliorate deficits in habit learning and spatial memory by modulating the dopaminergic neuronal and N-methyl-D-aspartate receptor-mediated signaling systems in MPTP-lesioned mice treated with L-DOPA. GPS may serve as an adjuvant therapeutic agent for memory deficits in patients with PD receiving L-DOPA.

This study investigated the effects of asarinin on dopamine biosynthesis and 6-hydroxydopamine (6-OHDA)-induced cytotoxicity in rat adrenal pheochromocytoma (PC12) cells. Treatment with asarinin (25-50 μM) increased intracellular dopamine levels and enhanced L-DOPA-induced increases in dopamine levels. Asarinin (25 μM) induced cyclic AMP-dependent protein kinase A (PKA) signaling, leading to increased cyclic AMP-response element binding protein (CREB) and tyrosine hydroxylase (TH) phosphorylation, which in turn stimulated dopamine production. Asarinin (25 μM) also activated transient phosphorylation of extracellular signal-regulated kinase (ERK1/2) and Bad phosphorylation at Ser 112, both of which have been shown to promote cell survival. In contrast, asarinin (25 μM) inhibited sustained ERK1/2, Bax, c-Jun N-terminal kinase (JNK1/2) and p38 mitogen-activated protein kinase (p38MAPK) phosphorylation and caspase-3 activity, which were induced by 6-OHDA (100 μM). These results suggest that asarinin induces dopamine biosynthesis via activation of the PKA-CREB-TH system and protects against 6-OHDA-induced cytotoxicity by inhibiting the sustained activation of the ERK-p38MAPK-JNK1/2-caspase-3 system in PC12 cells.

This study investigated the effects of ethanol extract from Gynostemma pentaphyllum (GP-EX) on memory deficits in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned mouse model of Parkinson's disease (PD) (MPTP-lesioned mice). MPTP (30 mg/kg/day, 5 days)-lesioned mice showed deficits of habit learning memory and spatial memory, which were further aggravated by treatment with L-3,4-dihydroxyphenylalanine (L-DOPA) (25 mg/kg, 21 days). However, treatment with GP-EX (50 mg/kg, 21 days) ameliorated memory deficits in MPTP-lesioned mice treated with L-DOPA (25 mg/kg): GP-EX prevented the decreases in retention latency time in the passive avoidance test and tyrosine hydroxylase-immunopositive cells and dopamine levels in the nigrostriatum. GP-EX also reduced increases in retention transfer latency time of the elevated plus-maze test and expression of N-methyl-D-aspartate (NMDA) receptor and improved decreases in phosphorylation of extracellular signal-regulated kinase (ERK1/2) and cyclic AMP-response element binding protein (CREB) in the hippocampus in the same models. By contrast, L-DOPA treatment (10 mg/kg, 21 days) ameliorated memory deficits in MPTP-lesioned mice, which were further improved by GP-EX treatment. These results suggest that GP-EX ameliorates habit learning memory deficits by activating dopaminergic neurons and spatial memory deficits by modulating NMDA receptor-ERK1/2-CREB system in MPTP-lesioned mice treated with L-DOPA. GP-EX may serve as an adjuvant phytonutrient for memory deficits in PD.

This study investigated the effects of (-)-sesamin on chronic electric footshock (EF) stress-induced anxiety disorders in mice. Mice were treated with (-)-sesamin (25 and 50 mg/kg) orally once a day for 21 days prior to exposure to EF stress (0.6 mA, 1 s every 5 s, 3 min). Mice treated with (-)-sesamin (25 and 50 mg/kg) exhibited less severe decreases in the number of open arm entries and time spent on open arms in the elevated plus-maze test and the distance traveled in the open field test following exposure to chronic EF stress. Similarly, mice treated with (-)-sesamin exhibited significantly less severe decreases in brain levels of dopamine, norepinephrine, and serotonin following exposure to chronic EF stress. Increases in serum levels of corticosterone and expression of c-Fos were also less pronounced in mice treated with (-)-sesamin (25 and 50 mg/kg). These results suggest that (-)-sesamin may protect against the effects of chronic EF stress-induced anxiety disorders by modulating dopamine, norepinephrine, and serotonin levels, c-Fos expression, and corticosterone levels.

This study investigated the effects of (-)-sesamin on memory deficits induced by chronic electric footshock (EF)-induced stress in mice. Mice were treated with (-)-sesamin (25 and 50mg/kg, p.o., daily for 21day) prior to chronic EF stress (0.6mA, 1s every 5s for 3min, daily for 21day). Transfer retention latencies in the elevated plus maze test and N-methyl-d-aspartate (NMDA) receptor (type 1) phosphorylation in the hippocampus increased with chronic EF stress, and they were reduced by treatment with (-)-sesamin at both doses. Phosphorylation of extracellular signal-regulated kinase (ERK1/2) and cyclic AMP-responsive element binding protein (CREB), which were reduced by chronic EF stress, were increased by treatment with (-)-sesamin. Retention latencies in the passive avoidance test and dopamine levels in the substantia nigra-striatum were also reduced by chronic EF stress, and similarly recovered with (-)-sesamin treatment. These results suggest that (-)-sesamin ameliorates the effects of chronic EF stress-induced spatial and habit learning memory deficits by modulating both NMDA receptor and dopaminergic neuronal systems.

Gynostemma (G.) pentaphyllum (Cucurbitaceae) contains various bioactive gypenosides. Ethanol extract from G. pentaphyllum (GP-EX) has been shown to have ameliorative effects on the death of dopaminergic neurons in animal models of Parkinson’s disease (PD) induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine- and 6-hydroxydopamine. PD patients exhibit multiple symptoms, so PD-related research should combine neurotoxin models with genetic models. In the present study, we investigated the ameliorative effects of GP-EX, including gypenosides, on the cell death of dopaminergic neurons in the midbrain of A53T α-synuclein transgenic mouse models of PD (A53T). Both GP-EX and gypenosides at 50 mg/kg per day were orally administered to the A53T mice for 20 weeks. α-Synuclein-immunopositive cells and α-synuclein phosphorylation were increased in the midbrain of A53T mice, which was reduced following treatment with GP-EX. Treatment with GP-EX modulated the reduced phosphorylation of tyrosine hydroxylase, extracellular signal-regulated kinase (ERK1/2), Bcl-2-associated death promoter (Bad) at Ser112, and c-Jun N-terminal kinase (JNK1/2) due to α-synuclein overexpression. In the A53T group, GP-EX treatment prolonged the latency of the step-through passive avoidance test and shortened the transfer latency of the elevated plus maze test. Gypenosides treatment exhibited the effects and efficacy similar to those of GP-EX. Taken together, GP-EX, including gypenosides, has ameliorative effects on dopaminergic neuronal cell death due to the overexpression of α-synuclein by modulating ERK1/2, Bad at Ser112, and JNK1/2 signaling in the midbrain of A53T mouse model of PD. Further studies are needed to investigate GP-EX as a treatment for neurodegenerative synucleinopathies, including PD. This study was approved by the Animal Ethics Committee of Chungbuk National University (approval No. CBNUA-956-16-01) on September 21, 2016.

The aim of this study was to evaluate the appropriateness and clinical outcomes of edoxaban use, and to determine the role of clinical pharmacists in improving the efficacy and safety of edoxaban use.A retrospective study was performed by using an electronic medical record and anticoagulation clinical data from 600 patients who received edoxaban from 1 March 2016 to 16 July 2017 at a tertiary teaching university hospital. The appropriateness of edoxaban use was assessed using eight criteria based on drug use evaluation criteria developed by the American Society of Health-System Pharmacists drug use evaluation guidelines, details in Korea Food and Drug Administration approval of edoxaban. Clinical outcomes were evaluated between the appropriately prescribed and inappropriately prescribed groups regarding the incidence of thrombosis and bleeding episodes.After excluding 86 patients due to the inability to assess renal function, 514 were eligible. Appropriate use was found in 294 patients (57.2%). The most frequent inappropriate use of edoxaban was dose adjustment (60.8%) in accordance with the dosing recommendation in patients with renal insufficiency (creatinine clearance [CrCl] of 15-50 mL/min) and a low body weight of60 kg. Moreover, there were three cases of edoxaban use in patients with prosthetic heart valves and moderate-to-severe mitral stenosis, and 15 cases of non-valvular atrial fibrillation in patients with CrCl95 mL/min in whom edoxaban use is not recommended. Furthermore, we found that the factors related to the appropriateness of edoxaban use were60 kg body weight (adjusted odds ratio [OR]: 0.310; confidence interval [CI]: 0.197-0.488) and CrCl50 mL/min (adjusted OR: 0.629; CI: 0.404-0.980). There were 45 events (8.75%) of any bleeding, 9 (1.8%) of stroke/transient ischaemic attack (TIA) and four events (0.8%) of deep vein thrombosis (DVT)/pulmonary embolism (PE). However, there was no difference between the appropriately prescribed group (294 patients) and inappropriately prescribed group (220 patients) in the incidence of bleeding events (27 [9.2%] vs 18 [8.2%]), stroke/TIA (7 [2.4%] vs 2 [0.9%]) and DVT/PE (2 [0.7%] vs 2 [0.9%]), respectively.Although edoxaban has a broad therapeutic window that does not require routine monitoring, it should be cautiously used in patients with renal insufficiency (CrCl50 mL/min) and body weight60 kg.

1-O-Hexyl-2,3,5-trimethylhydroquinone (HTHQ) has previously been found to have effective anti-oxidant and anti-lipid-peroxidative activity. We aimed to elucidate whether HTHQ can prevent dopaminergic neuronal cell death by investigating the effect on l-DOPA-induced cytotoxicity in PC12 cells. HTHQ protected from both l-DOPA-induced cell death and superoxide dismutase activity reduction. When assessing the effect of HTHQ on oxidative stress-related signaling pathways, HTHQ inhibited l-DOPA-induced phosphorylation of sustained extracellular signal-regulated kinases (ERK1/2), p38 mitogen-activated protein kinase (MAPK), and c-Jun N-terminal kinase (JNK1/2). HTHQ also normalized l-DOPA-reduced Bcl-2-associated death protein (Bad) phosphorylation and Bcl-2-associated X protein (Bax) expression, promoting cell survival. Taken together, HTHQ exhibits protective effects against l-DOPA-induced cell death through modulation of the ERK1/2-p38MAPK-JNK1/2-Bad-Bax signaling pathway in PC12 cells. These results suggest that HTHQ may show ameliorative effects against oxidative stress-induced dopaminergic neuronal cell death, although further studies in animal models of Parkinson&rsquo, s disease are required to confirm this.

We investigated the antihyperglycemic and antioxidant activities of polysaccharides produced from Pleurotus ferulae mutant in streptozotocin-induced diabetic rats. Blood glucose level in the STZ-induced diabetic rat administered the extract polysaccharides, 250mg/kg b·w/d (EPSG) was 196.97mg/dL, approximately 54.12% less compared to that of the STZ-induced diabetic rats administered 0.9% NaCl solution (NCG). The insulin level in the EPSG was approximately 1.64-fold higher than that in the NCG. HDL and LDL cholesterol levels in the EPSG were 29.15mg/L and 20.35mg/dL, respectively, representing an approximate increase of 69.18% and decrease of 38.52%, respectively. The activities of aspartate aminotransferase (AST) and alanine transferase (ALT) in the EPSG decreased approximately by 49.27 and 50.43%, respectively, while the alkaline phosphatase (ALP) activity decreased by 34.25%, relative to the NCG. Antioxidant activities in the NCG decreased relative to the normal group. In contrast, for EPSG, these values increased relative to the NCG. The malondialdehyde level in the EPSG was 12.95mmol/mg protein, which was approximately 70.64% of that in the NCG. These results suggest that the polysaccharides of Pleurotus ferulae mutant could be developed as potential antidiabetic agents or functional foods for people with a high risk of diabetes mellitus.

Ginseng has a long history of use as a tonic for restoration of vigor. One example of ginseng-derived tonic effect is that it can improve physical stamina under conditions of stress. However, the active ingredient and the underlying molecular mechanism responsible for the ergogenic effect are unknown. Recent studies show that ginseng contains a novel ingredient, gintonin, which consists of a unique class of herbal-medicine lysophosphatidic acids (LPAs). Gintonin activates G protein-coupled LPA receptors to produce a transient [Ca(2+)]i signal, which is coupled to diverse intra- and inter-cellular signal transduction pathways that stimulate hormone or neurotransmitter release. However, relatively little is known about how gintonin-mediated cellular modulation is linked to physical endurance. In the present study, systemic administration of gintonin, but not ginsenosides, in fasted mice increased blood glucose concentrations in a dose-dependent manner. Gintonin treatment elevated blood glucose to a maximum level after 30min. This elevation in blood glucose level could be abrogated by the LPA1/3 receptor antagonist, Ki16425, or the β-adrenergic receptor antagonist, propranolol. Furthermore, gintonin-dependent enhanced performance of fasted mice in rotarod test was likewise abrogated by Ki16425. Gintonin also elevated plasma epinephrine and norepinephrine concentrations. The present study shows that gintonin mediates catecholamine release through activation of the LPA receptor and that activation of the β-adrenergic receptor is coupled to liver glycogenolysis, thereby increasing the supply of glucose and enhancing performance in the rotarod test. Thus, gintonin acts via the LPA-catecholamine-glycogenolysis axis, representing a candidate mechanism that can explain how ginseng treatment enhances physical stamina.

Gynostemma (G.) pentaphyllum (Cucurbitaceae) contains various bioactive gypenosides. Ethanol extract from G. pentaphyllum (GP-EX) has been shown to have ameliorative effects on the death of dopaminergic neurons in animal models of Parkinson’s disease (PD) induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine- and 6-hydroxydopamine. PD patients exhibit multiple symptoms, so PD-related research should combine neurotoxin models with genetic models. In the present study, we investigated the ameliorative effects of GP-EX, including gypenosides, on the cell death of dopaminergic neurons in the midbrain of A53T α-synuclein transgenic mouse models of PD (A53T). Both GP-EX and gypenosides at 50 mg/kg per day were orally administered to the A53T mice for 20 weeks. α-Synuclein-immunopositive cells and α-synuclein phosphorylation were increased in the midbrain of A53T mice, which was reduced following treatment with GP-EX. Treatment with GP-EX modulated the reduced phosphorylation of tyrosine hydroxylase, extracellular signal-regulated kinase (ERK1/2), Bcl-2-associated death promoter (Bad) at Ser112, and c-Jun N-terminal kinase (JNK1/2) due to α-synuclein overexpression. In the A53T group, GP-EX treatment prolonged the latency of the step-through passive avoidance test and shortened the transfer latency of the elevated plus maze test. Gypenosides treatment exhibited the effects and efficacy similar to those of GP-EX. Taken together, GP-EX, including gypenosides, has ameliorative effects on dopaminergic neuronal cell death due to the overexpression of α-synuclein by modulating ERK1/2, Bad at Ser112, and JNK1/2 signaling in the midbrain of A53T mouse model of PD. Further studies are needed to investigate GP-EX as a treatment for neurodegenerative synucleinopathies, including PD. This study was approved by the Animal Ethics Committee of Chungbuk National University (approval No. CBNUA-956-16-01) on September 21, 2016.

This study investigated the effects of ombuoside on L-3,4-dihydroxyphenylalanine (L-DOPA)-induced neurotoxicity in PC12 cells. Ombuoside did not affect cell viability at concentrations of up to 50 µM for 24 h, and ombuoside (1, 5, and 10 µM) significantly inhibited L-DOPA-induced (100 and 200 µM) decreases in cell viability. L-DOPA (100 and 200 µM) induced sustained phosphorylation of extracellular signal-regulated kinases (ERK1/2) for 6 h, which were significantly decreased by cotreatments with ombuoside (1, 5, and 10 µM). L-DOPA (100 and 200 µM) alone significantly increased c-Jun N-terminal kinase (JNK1/2) phosphorylation for 6 h and cleaved-caspase-3 expression for 24 h, both of which were partially, but significantly, blocked by ombuoside (1, 5, and 10 µM). In addition, ombuoside (1, 5, and 10 µM) significantly restored the L-DOPA-induced (100 and 200 µM) decrease in superoxide dismutase (SOD) activity for 24 h. Taken together, these findings indicate that ombuoside protects against L-DOPA-induced neurotoxicity by inhibiting L-DOPA-induced increases in sustained ERK1/2 and JNK1/2 phosphorylation and caspase-3 expression and L-DOPA-induced decrease in SOD activity in PC12 cells. Thus, ombuoside might represent a novel neuroprotective agent that warrants further study.

Five new dammarane-type saponins, gypenosides GD1-GD5 (1-5), along with six known saponins (6-11), were isolated from the aerial parts of Gynostemma pentaphyllum using various chromatographic methods. Their structures were elucidated by a combination of spectroscopic and spectrometric data, including 1D and 2D NMR and HRESIMS. All isolates were tested for their inhibitory effects on IL-6-induced STAT3 promoter activity in Hep3B cells. Compounds 1, 9, and 11 displayed potent inhibitory effects, with IC50 values ranging from 0.27 to 0.59 μM.

The present study investigated the effects of (-)-sesamin on 6-hydroxydopamine (6-OHDA)-induced neurotoxicity using PC12 cells and dopaminergic neuronal cells of 6-OHDA-lesioned rat model of Parkinson's disease (PD). In PC12 cells, treatment with (-)-sesamin (25 µM) reduced 6-OHDA (100 µM)-induced cell death and induced transient extracellular signal-regulated kinase (ERK1/2) phosphorylation and Bad phosphorylation at Ser112 (BadSer112). In contrast, sustained ERK1/2 phosphorylation, p38 mitogen-activated protein kinase (p38MAPK) and c-Jun N-terminal kinase (JNK1/2) phosphorylation, and cleaved-caspase-3 activity, all of which were induced by 6-OHDA (100 µM), were inhibited by treatment with (-)-sesamin (25 µM). Furthermore, co-treatment with (-)-sesamin (30 mg/kg, p.o.) once a day for 28 days significantly increased the number of tyrosine hydroxylase-immunopositive neuronal cells and the levels of dopamine, norepinephrine, 3,4-dihydroxyphenylacetic acid, and homovanillic acid in the substantia nigra-striatum of 6-OHDA-lesioned rat model of PD with or without L-DOPA treatment. These results suggest that (-)-sesamin protects 6-OHDA-induced cytotoxicity via the activation of transient ERK1/2-BadSer112 system and the inhibition of sustained ERK-p38MAPK-JNK1/2-caspase-3 system in PC12 cells. (-)-Sesamin also shows protective effects on long-term L-DOPA therapy in dopaminergic neuronal cells of PD rat models. (-)-Sesamin may serve as adjuvant therapeutics in PD.

Gintonin is a novel ginseng-derived G protein-coupled lysophosphatidic acid (LPA) receptor ligand. Gintonin elicits an intracellular calcium concentration [Ca(2+)]i transient via activation of LPA receptors and regulates calcium-dependent ion channels and receptors. [Ca(2+)]i elevation by neurotransmitters or depolarization is usually coupled to neurotransmitter release in neuronal cells. Little is known about whether gintonin-mediated [Ca(2+)]i transients are also coupled to neurotransmitter release. The PC12 cell line is derived from a pheochromocytoma of the rat adrenal medulla and is widely used as a model for catecholamine release. In the present study, we examined the effects of gintonin on dopamine release in PC12 cells. Application of gintonin to PC12 cells induced [Ca(2+)]i transients in concentration-dependent and reversible manners. However, ginsenoside Rg3, another active ingredient of ginseng, induced a lagged and irreversible [Ca(2+)]i increase. The induction of gintonin-mediated [Ca(2+)]i transients was attenuated or blocked by the LPA1/3 receptor antagonist Ki16425, a phospholipase C inhibitor, an inositol 1,4,5-triphosphate receptor antagonist, and an intracellular Ca(2+) chelator. Repeated treatment with gintonin induced homologous desensitization of [Ca(2+)]i transients. Gintonin treatment in PC12 cells increased the release of dopamine in a concentration-dependent manner. Intraperitoneal administration of gintonin to mice also increased serum dopamine concentrations. The present study shows that gintonin-mediated [Ca(2+)]i transients are coupled to dopamine release via LPA receptor activation. Finally, gintonin-mediated [Ca(2+)]i transients and dopamine release via LPA receptor activation might explain one mechanism of gintonin-mediated inter-neuronal modulation in the nervous system.

We investigated the physicochemical properties, chemical composition, stability and antioxidant activity from seed oil of Salicornia herbaciea grown in Korea. The density, refractive index, acid value, peroxide value, iodine value, saponification value, and unsaponifiable matter of oil were 0.91mg/mL, 1.48 at 20 °C, 1.89mg KOH/g oil, 10.20 mEq/kg oil, 1.08 g I/g oil, 216.21 mg KOH/g oil, and 2.60%, respectively. The major fatty acids were linoleic acid (43.73%), oleic acid (19.81%), arachidic acid (13.52%), and palmitic acid (11.84%), respectively. The oil contained high levels of α-tocopherol (249.2 mg/kg oil), followed by δ-tocopherol (89.3 mg/kg), and γ-tocopherol (75.6 mg/kg oil). The oil was found to have high levels of β-sitosterol (94.5mg/kg oil) and stigmasterol (65.7mg/kg oil), respectively. The total phenol, chlorophyll and β-carotene content of oil was 15.2, 94.5, and 8.2 mg/kg oil, respectively. The oil had good oxidative stability during 60 days of storage in a dark area at 50 °C. The maximum degradation rates of the oil were observed at 242.3 °C (9.5%/min), 382.6 °C (5.2%/min), and 440.7 °C (1.3%/min), respectively, where the rate of the weight decrease increased to a maximum up to this point. The ABTS radical scavenging activity of the oil was increased from 50.2 to 71.8% when the oil concentration extracted by methanol was increased from 100 to 300 μg/mL. This study suggests that S. herbaciea seed oil has potential use in functional foods, cosmetics or pharmaceuticals.

The effects of berberine on long-term administration of L-DOPA in 6-hydroxydopamine (6-OHDA)-lesioned rat model of Parkinson's disease (PD) were investigated. Rat models of PD were prepared by 6-OHDA lesions in the ipsilateral sides, and then were treated with berberine (5 and 15 mg/kg) and/or L-DOPA (10 mg/kg) once daily for 21 days. Treatments with either concentration of berberine (5 and 15 mg/kg) in 6-OHDA-lesioned groups decreased the numbers of tyrosine hydroxylase (TH)-immunopositive neurons in the substantia nigra and the levels of dopamine, norepinephrine, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in the striatum as compared to 6-OHDA-lesioned groups. In addition, dopaminergic neuronal cell death of the ipsilateral sides in 6-OHDA-lesioned groups was attenuated by L-DOPA administration. However, both concentrations of berberine in 6-OHDA-lesioned groups treated with L-DOPA aggravated the numbers of TH-immunopositive neurons in the substantia nigra and the levels of dopamine, norepinephrine, DOPAC and HVA in the striatum as compared to rats not treated with berberine. These results suggest that berberine leads to the degeneration of dopaminergic neuronal cells in the substantia nigra in the rat model of PD with chronic L-DOPA administration. Long-term L-DOPA therapy that may involve possibly neurotoxic isoquinoline agents including berberine should involve monitoring for adverse symptoms.

We investigated the antioxidant, nitrite scavenging, immune-enhancing and antimicrobial effects of the Lentinus edodes fruiting body extracts (LEFB extract) cultivated in the solid state using corn cob. The CAT, SOD and GSH-Px activities in the LEFB extract (400mg)-fed group were approximately 1.70, 1.64, and 2.2 times higher than those of the control group. The nitrite scavenging activity was highest (86.4%) when the LEFB extract concentration was 600 μg/mL. The immune-enhancing and antimicrobial effects of LEFB extract after an experimental Bordetella bronchiseptica infection were examined. White blood and neutrophil in the LEFB extract (400 mg)-fed group were approximately 2.1 and 2.2 times higher, respectively, than those of the control group. The total leukocyte was 30% higher than that of the control group. The CD4: CD8 ratio in the LEFB extract (200 mg)-fed group was increased approximately 54.3% compared to that of the control group. The levels of TNF-α and IFN-γ mRNA expression in the LEFB extract (400 mg)-fed group were approximately 49 and 50% lower than those of the control group. The bacteria and inflammatory cell in the LEFB extract (400 mg)-fed group were approximately 48.7 and 29% lower, respectively, than that of the control group. These results suggest that the LEFB extract may be useful in immune function improvement.

Constitutive expression of C–C chemokine receptor (CCR) 5 has been detected in astrocytes, microglia and neurons, but its physiological roles in the central nervous system are obscure. The bidirectional interactions between neuron and glial cells through CCR5 and its ligands were thought to be crucial for maintaining normal neuronal activities. No study has described function of CCR5 in the dopaminergic neurodegeneration in Parkinson's disease. In order to examine effects of CCR5 on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced dopaminergic neurodegeneration, we employed CCR5 wild type (WT) and knockout (KO) mice. Immunostainings for tyrosine hydroxylase (TH) exhibited that CCR5 KO mice had lower number of TH-positive neurons even in the absence of MPTP. Difference in MPTP (15 mg/kg×4 times, 2 hr interval)-mediated loss of TH-positive neurons was subtle between CCR5 WT and KO mice, but there was larger dopamine depletion, behavioral impairments and microglial activation in CCR5 deficient mice. Intriguingly, CCR5 KO brains contained higher immunoreactivity for monoamine oxidase (MAO) B which was mainly localized within astrocytes. In agreement with upregulation of MAO B, concentration of MPP+ was higher in the substantia nigra and striatum of CCR5 KO mice after MPTP injection. We found remarkable activation of p38 MAPK in CCR5 deficient mice, which positively regulates MAO B expression. These results indicate that CCR5 deficiency modifies the nigrostriatal dopaminergic neuronal system and bidirectional interaction between neurons and glial cells via CCR5 might be important for dopaminergic neuronal survival.

The phytochemical investigation of the aerial parts of Gynostemma pentaphyllum led to the isolation of a new flavonol glycoside, gynopentaphylloside (1), along with seven known compounds (2-8). The structure of the new compound was determined on the basis of 1D, 2D NMR and HRESIMS data as well as acid hydrolysis. The antioxidant activity of the isolates was evaluated by a 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay.

The immunomodulatory effects of the ethanol extract of Gynostemma pentaphyllum (GP-EX) were examined in electric footshock (EFS)-stressed mice. The mice were orally administered various doses of GP-EX for 7 days before exposure to EFS (duration: 3 min, interval: 10 s, intensity: 2 mA) once a day from day 8 for 14 days with continuous daily feeding of GP-EX. Oral administration of GP-EX to mice prevented EFS stress-induced immunosuppression as determined by the lymphoid organ (thymus and spleen) weight and cellularity. In addition, oral administration of GP-EX restored EFS-suppressed functional properties of mature lymphocytes in terms of concanavalin A-induced proliferation of splenocytes and lipopolysaccharide-induced cytokine production (TNF-α, IL-1β). Furthermore, we found that mice that were orally administered with GP-EX generated much more potent ovalbumin-specific cytotoxic T lymphocyte responses upon intravenous ovalbumin injection compared to the untreated controls. These results demonstrate that oral administration of the ethanol extract of Gynostemma pentaphyllum could increase host defense in immunocompromised situations such as stress-induced immunosuppression.

The intracellular levels of cyclic AMP (cAMP) increase in response to cytotoxic concentrations of L-DOPA in PC12 cells, and forskolin that induces intracellular cAMP levels either protects PC12 cells from L-DOPA-induced cytotoxicity or enhances cytotoxicity in a concentration-dependent manner. This study investigated the effects of cAMP induced by forskolin on cell viability of PC12 cells, relevant to L-DOPA-induced cytotoxicity in Parkinson's disease therapy. The low levels of forskolin (0.01 and 0.1 μM)-induced cAMP increased dopamine biosynthesis and tyrosine hydroxylase (TH) phosphorylation, and induced transient phosphorylation of ERK1/2 within 1 h. However, at the high levels of forskolin (1.0 and 10 μM)-induced cAMP, dopamine biosynthesis and TH phosphorylation did not increase, but rapid differentiation in neurite-like formation was observed with a steady state. The high levels of forskolin-induced cAMP also induced sustained increase in ERK1/2 phosphorylation within 0.25-6 h and then led to apoptosis, which was apparently mediated by JNK1/2 and caspase-3 activation. Multiple treatment of PC12 cells with nontoxic L-DOPA (20 μM) for 4-6 days induced neurite-like formation and decreased intracellular dopamine levels by reducing TH phosphorylation. These results suggest that the low levels of forskolin-induced cAMP increased dopamine biosynthesis in cell survival via transient ERK1/2 phosphorylation. In contrast, the high levels of forskolin-induced cAMP induced differentiation via sustained ERK1/2 phosphorylation and then led to apoptosis. Taken together, the intracellular levels of cAMP play a dual role in cell survival and death through the ERK1/2 and JNK1/2 pathways in PC12 cells.

We investigated the effects of the antioxidant and the nitrite scavenging activities of the extracts from Pleurotus ferulae fruiting body grown on the solid state using corn cob and activated bleaching earth (CCABE media) and its mycelium grown in the liquid state. The total phenol and polysaccharide concentrations in hot water extract of fruiting body were approximately 3.6- and 4.3-fold higher than those of the mycelium. Using the hot water extract of fruiting body, the maximum DPPH radical scavenging activity at 9 mg/mL, hydroxyl radical scavenging activity at 12mg/mL, reducing power at 12 mg/mL, and chelating ability at 12 mg/mL were obtained, 80.5%, 72.4%, 0.99 OD (700 nm), and 77.0%, respectively. However, in the case of hydrogen peroxide scavenging activity, the ethanol extract was the highest, 78.7% at 12 mg/mL. The maximum nitrite scavenging activity was obtained, 89.7% at 6 mg/mL of hot water extract from fruiting body. Hot water extracts were more effective than ethanol extracts in scavenging activity on DPPH radicals and hydroxyl radical scavenging, reducing power, and chelating activity of ferrous, whereas ethanol extracts were more effective in hydrogen peroxide scavenging activity as evidenced by their lower EC50 values. These results indicate that the hot water extract of P. ferulae fruiting body using CCABE media has good potential to be used as a source of materials or additives for oxidation suppressant in food, cosmetics and drug compositions.

The effects of a water-soluble tacrolimus-PEG conjugate (KI-102) on insulin-dependent diabetes mellitus and systemic lupus erythematosus were investigated. KI-102 was stable at pH 4.0-4.5 and 4°C. The area under the concentration-time curve, the time of maximum concentration, and the maximum concentration were 43.4 ng·h/mL, 0.85 h, and 8.1 ng/mL, respectively, similar to those of FK506. Mice that administered KI-102 at 4.32 mg/kg had the plasma glucose concentrations that decreased to 7.5 mmol/L after 170 days, similar to that of mice administered FK506 at 0.6 mg/kg. There were no incidences of diabetes when KI-102 was administered at 86.4 mg/kg after 24 weeks. The group that administered 43.2 mg/kg had decreases in the concentrations of β-hydroxybutyrate (60%), triglyceride (24%), and cholesterol (30%). KI-102 administered at 180 mg/kg reduced serum anti-dsDNA antibody activity by 64% compared with a control. Urinary albumin concentration in the same group decreased 81% compared with the control. These results indicate that KI-102 may be practically applicable as prodrug of FK506.

We determined the optimal culture and medium conditions for effective production of mycelial mass and exopolysaccharide from a liquid culture of Mycoleptodonoides aitchisonii in an air-lift bioreactor. The mycelial growth and exopolysaccharide production were found to be optimal at a temperature of 25 °C and pH of 6.5. When 60 g/L of lactose was used as a carbon source, the maximum mycelial growth and exopolysaccharide production were obtained. The polypeptone and yeast extract were the most appropriate nitrogen sources for mycelial growth and exopolysaccharide production. In addition, when a mixture of 20 g/L of polypeptone and 5 g/L of yeast extract was used, the exopolysaccharide production increased 50% compared to that of the sole nitrogen source. CaCl2·2H2O (1.0 g/L) was the most effective mineral source. Using the optimal culture and medium conditions, batch cultures with basal and designed medium on mycelial growth and exopolysaccharide production in a 5 L air-lift bioreactor were carried out for 16 days. The mycelial growth and exopolysaccharide production increased with an increase of culture time at 14 days, and the maximum mycelial growth and exopolysaccharide production were 20.3 and 6.2 g/L, respectively, after 14 days of culture. The developed model in an air-lift bioreactor showed good agreement with experimental data. These results indicate that exopolysaccharide production is associated with the mycelial growth of M. aitchisonii in an air-lift bioreactor.

L-DOPA therapy for Parkinson's disease has a double-edge effect on nigrostriatal dopaminergic neurons: L-DOPA increases the intracellular level of dopamine, but it induces neuron cytotoxicity in a concentration-dependent manner. To investigate the molecular signaling mechanisms that underlie the concentration-dependent effects of L-DOPA on cell viability, the activities of mitogen-activated protein kinases (MAPKs) and apoptotic enzymes were measured in rat adrenal pheochromocytoma (PC12) cells in the presence of a low concentration (20 μM) and high concentrations (100–200 μM) of L-DOPA. At the low concentration, L-DOPA was not cytotoxic and its presence increased the activities of extracellular signal-regulated kinase (ERK)1/2, p38 MAPK, BadSer112, Bcl-2, and caspase-12. At the high concentrations, L-DOPA was cytotoxic and stimulated the activities of ERK1/2, p38 MAPK, c-Jun N-terminal kinase (JNK)1/2, BadSer155, caspase-12 and caspase-3. The increased levels of ERK1/2 and BadSer155 in the presence of high concentrations of L-DOPA did not protect against L-DOPA-mediated cytotoxicity. In addition, the levels of L-type Ca2+ channel-sensitive intracellular cyclic AMP (cAMP) and Ca2+ were elevated in the presence of L-DOPA, and the increase in the levels of intracellular cAMP may also play a role in cellular viability, since cAMP levels and cytotoxicity increased in parallel with L-DOPA concentrations and the addition of forskolin in the medium increased cytotoxicity in a concentration-dependent manner. These results suggest that, at a low and non-toxic concentration, L-DOPA may promote cell survival by increasing the activities of ERK1/2, BadSer112 and Bcl-2, while, at high concentrations, L-DOPA activates the caspase-3 cell death enzyme through the JNK1/2 and p38 MAPK signaling pathways as well as endoplasmic reticulum stress that activates caspase-12. Intracellular cAMP levels may also play a role here. The results may lead to an effective therapy for Parkinson's disease.

The effects of scoparone on dopamine release in PC12 cells were investigated. Scoparone at 50-200 microM increased dopamine release into the culture medium. However, the released levels of dopamine by scoparone were not altered in the absence of extracellular Ca(2+) and by adenylyl cyclase inhibitor MDL-12,330A. Scoparone increased phosphorylation of PKA, CaMK II and synapsin I. Scoparone also enhanced K(+)-induced levels of dopamine release by CaMK II phosphorylation. These results suggest that scoparone increases dopamine release by synapsin I phosphorylation via activation of PKA and CaMK II, which are mediated by cyclic AMP levels and Ca(2+) influx.

6-Hydroxydopamine administration for 28 days (8 microg/2 microL) reduced the number of tyrosine hydroxylase (TH)-immunopositive neurons to 40.2% in the substantia nigra compared to the intact contralateral side. Dopamine, 3,4-dihydroxyphenylacetic acid, homovanillic acid and norepinephrine levels were reduced to 19.1%, 52.3%, 47.1% and 67.4% in the striatum of 6-hydroxydopamine-lesioned rats compared to the control group, respectively. However, an oral administration of herbal ethanol extracts from Gynostemma pentaphyllum (GP-EX) (10 mg/kg and 30 mg/kg) starting on day 3 post-lesion for 28 days markedly ameliorated the reduction of TH-immunopositive neurons induced by 6-hydroxydopamine-lesioned rat brain from 40.2% to 67.4% and 75.8% in the substantia nigra. GP-EX administration (10 and 30 mg/kg) also recovered the levels of dopamine, 3,4-dihydroxyphenylacetic acid, homovanillic acid and norepinephrine in post-lesion striatum to 64.1% and 65.0%, 77.9% and 89.7%, 82.6% and 90.2%, and 88.1% and 89.2% of the control group. GP-EX at the given doses did not produce any sign of toxicity such as weight loss, diarrhea and vomiting in rats during the 28 day treatment period and four gypenoside derivatives, gynosaponin TN-1, gynosaponin TN-2, gypenoside XLV and gypenoside LXXIV were identified from GP-EX. These results suggest that GP-EX might be helpful in the prevention of Parkinson's disease.

A highly selective and facile assay of human immunodeficiency virus protease (HIV-PR) has been required for the screening of medicinal inhibitors and also for classifying the subtypes of HIV in the therapeutic treatment of acquired immune deficiency syndrome (AIDS). This article describes a novel assay method of HIV-PR based on the selective fluorogenic reaction of peptides. A peptide fragment generated from a substrate by the enzymatic digestion with HIV-PR could be selectively quantified by the spectrofluorometric detection after the fluorogenic reaction with catechol in the presence of sodium periodate and sodium borate (pH 7.0). This assay system uses an N-terminal acetyl peptide as the substrate and crude extracts from Escherichia coli expressing recombinant HIV-PR. The activity obtained by the proposed assay correlated with that obtained by a conventional HIV-PR assay based on fluorescence resonance energy transfer detection.

The effects of catalponol (1) on dopamine biosynthesis and L-DOPA-induced cytotoxicity in PC12 cells were investigated. Catalponol at concentration ranges of 1-5 microM increased the intracellular levels of dopamine at 12-48 h. Catalponol at concentrations of up to 10 microM did not alter cell viability. Tyrosine hydroxylase (TH) activity was enhanced by 1 at 3 microM in a time-dependent manner, but aromatic L-amino acid decarboxylase activity was not. Catalponol also increased the intracellular levels of cyclic AMP and TH phosphorylation. In addition, catalponol at 3 microM associated with L-DOPA (20-50 microM) further enhanced the increases in dopamine levels induced by L-DOPA (50-100 microM) at 24 h. Catalponol at 2-5 microM inhibited L-DOPA (100-200 microM)-induced cytotoxicity at 48 h. These results suggest that 1 enhanced dopamine biosynthesis by inducing TH activity and protected against L-DOPA-induced cytotoxicity in PC12 cells, which was mediated by the increased levels of cyclic AMP.

This experiment was performed to investigate whether methanol extract of Longanae Arillus (MELA) has hypnotic effects and/or enhances pentobarbital-induced sleep behaviors through the GABAergic systems. MELA prolonged sleep time and reduced sleep latency induced by pentobarbital similar to muscimol, a GABAA receptors agonist. MELA also increased sleep rate and sleep time in the combined administration with pentobarbital at the sub-hypnotic dosage and showed synergic effects with muscimol in potentiating sleep onset and enhancing sleep time induced by pentobarbital. However, MELA itself did not induce sleep at higher dose which was used in this experiment. In addition, both of MELA and pentobarbital increased chloride influx in primary cultured cerebellar granule cells. MELA increased GABAA receptors gamma-subunit expression and had no effect on the expression of alpha- and beta-subunits, and glutamic acid decarboxylase (GAD) in primary cultured cerebellar granule cells, showing different expression of subunits from pentobarbital. In conclusion, MELA itself does not induce sleep, but it augments pentobarbital-induced sleep behaviors through the modification of GABAergic systems.

The effects of asimilobine, an aporphine isoquinoline alkaloid, on dopamine biosynthesis and L-DOPA-induced cytotoxicity in PC12 cells were investigated. Asimilobine at concentration ranges of 0.05-0.2 microM showed a significant inhibition of intracellular dopamine levels for 24 h in a concentration-dependent manner with an IC50 value of 0.13 microM. Asimilobine at 0.15 microM inhibited tyrosine hydroxylase (TH) and aromatic L-amino acid decarboxylase (AADC) activities at 24 h (73.2% inhibition of TH activity): the inhibition of TH activity was stronger and longer than that of AADC activity. Asimilobine also decreased TH mRNA levels and intracellular cyclic AMP levels, but not the basal Ca2+ concentrations. In addition, asimilobine at 0.05-5.0 microM, but not 10 microM, did not alter cell viability toward PC12 cells. A non-cytotoxic asimilobine (0.15 microM) associated with l-DOPA (20, 50, and 100 microM) for 24 h inhibited L-DOPA-induced increases in dopamine levels and enhanced L-DOPA-induced cell death when compared with L-DOPA alone. These results suggest that asimilobine inhibits dopamine biosynthesis by mainly reducing the TH activity and TH mRNA expression, and enhances L-DOPA-induced cytotoxicity in PC12 cells.