(-)-Catechin-7-O-ß-d-Apiofuranoside Inhibits Hepatic Stellate Cell Activation by Suppressing the STAT3 Signaling Pathway.

Hepatic fibrosis is characterized by the abnormal deposition of extracellular matrix (ECM) proteins. During hepatic fibrogenesis, hepatic stellate cell (HSC) activation followed by chronic injuries is considered a key event in fibrogenesis, and activated HSCs are known to comprise approximately 90% of ECM-producing myofibroblasts. Here, we demonstrated that (-)-catechin-7-O-ß-d-apiofuranoside (C7A) significantly inhibited HSC activation via blocking the signal transducer and activator of transcription 3 (STAT3) signaling pathway. This is the first study to show the hepatic protective effects of C7A with possible mechanisms in vitro and in vivo. In our bioactivity screening, we figured out that the EtOH extract of Ulmusdavidiana var. japonica root barks, which have been used as a Korean traditional medicine, inhibited collagen synthesis in HSCs. Four catechins isolated from the EtOAc fraction of the EtOH extract were compared with each other in terms of reduction in collagen, which is considered as a marker of hepatic protective effects, and C7A showed the strongest inhibitory effects on HSC activation in protein and qPCR analyses. As a possible mechanism, we investigated the effects of C7A on the STAT3 signaling pathway, which is known to activate HSCs. We found that C7A inhibited phosphorylation of STAT3 and translocation of STAT3 to nucleus. C7A also inhibited expressions of MMP-2 and MMP-9, which are downstream genes of STAT3 signaling. Anti-fibrotic effects of C7A were evaluated in a thioacetamide (TAA)-induced liver fibrosis model, which indicated that C7A significantly inhibited ECM deposition through inhibiting STAT3 signaling. C7A decreased serum levels of aspartate amino transferase and alanine transaminase, which were markedly increased by TAA injection. Moreover, ECM-associated proteins and mRNA expression were strongly suppressed by C7A. Our study provides the experimental evidence that C7A has inhibitory effects on HSC activation after live injury and has preventive and therapeutic potentials for the management of hepatic fibrosis.

Suppression of 6-Hydroxydopamine-Induced Oxidative Stress by Hyperoside Via Activation of Nrf2/HO-1 Signaling in Dopaminergic Neurons.

In our ongoing research to discover natural products with neuroprotective effects, hyperoside (quercetin 3-O-galactoside) was isolated from Acer tegmentosum, which has been used in Korean traditional medicine to treat liver-related disorders. Here, we demonstrated that hyperoside protects cultured dopaminergic neurons from death via reactive oxygen species (ROS)-dependent mechanisms, although other relevant mechanisms of hyperoside activity remain largely uncharacterized. For the first time, we investigated the neuroprotective effects of hyperoside on 6-hydroxydopamine (6-OHDA)-induced neurotoxicity in neurons, and the possible underlying mechanisms. Hyperoside significantly ameliorated the loss of neuronal cell viability, lactate dehydrogenase release, excessive ROS accumulation and mitochondrial membrane potential dysfunction associated with 6-OHDA-induced neurotoxicity. Furthermore, hyperoside treatment activated the nuclear erythroid 2-related factor 2 (Nrf2), an upstream molecule of heme oxygenase-1 (HO-1). Hyperoside also induced the expression of HO-1, an antioxidant response gene. Remarkably, we found that the neuroprotective effects of hyperoside were weakened by an Nrf2 small interfering RNA, which blocked the ability of hyperoside to inhibit neuronal death, indicating the vital role of HO-1. Overall, we show that hyperoside, via the induction of Nrf2-dependent HO-1 activation, suppresses neuronal death caused by 6-OHDA-induced oxidative stress. Moreover, Nrf2-dependent HO-1 signaling activation represents a potential preventive and therapeutic target in Parkinson's disease management.

Determination of isoorientin levels in rat plasma after oral administration of Vaccinum bracteatum Thunb. methanol extract by high-performance liquid chromatography-tandem mass spectrometry.

A simple, sensitive and rapid liquid chromatography tandem mass spectrometry method (LC-MS/MS) was developed and validated for the determination of plasma isoorientin levels in rats. After simple protein precipitation using methanol, chromatographic analysis was performed using a Synergi 4µ polar-RP 80A column (150 × 2.0 mm, 4µm) under isocratic conditions and a mobile phase consisting of 0.1% formic acid in water and methanol (80:20, v/v) at a flow rate of 0.2 mL/min. In positive electrospray ionization mode, the protonated precursor and product ion transitions of isoorientin (m/z 449.0 → 299.1) and of puerarin (the internal standard; m/z 417.1 → 297.1) were acquired by multiple reaction monitoring. Calibration curves obtained for plasma showed good linearity over the concentration range 1-1000 ng/mL. The lower limit of quantification was 1 ng/mL. Intra- and inter-day precisions were within 8.8% relative standard deviation. Accuracies ranged from 92.1 and 109.7%. The isoorientin stability in rat plasma under typical handling/storage conditions also found to be acceptable. The developed method was applied successfully to a pharmacokinetic study of isoorientin orally administered as the methanol extract of Vaccinium bracteatum Thunb. or administered as pure isoorientin.

Liquiritigenin ameliorates memory and cognitive impairment through cholinergic and BDNF pathways in the mouse hippocampus.

Liquiritigenin (LQ), a flavonoid extracted from the radix of Glycyrrhiza, has anti-inflammatory and neuroprotective properties. In this study, we evaluated the cognitive enhancing effects of LQ on learning and memory impairments induced by scopolamine (0.5 mg/kg, i.p.), a muscarinic antagonist, using the Y-maze, passive avoidance, and novel object recognition tests. A single administration of LQ significantly improved scopolamine-induced cognitive impairments in these behavioral tests. In addition, LQ dramatically inhibited acetylcholinesterase and thiobarbituric acid reactive substance activities in the hippocampus of scopolamine-induced mice in a dose-dependent manner. Furthermore, LQ markedly increased the protein level of brain-derived neurotrophic factor (BDNF) and phosphorylation of extracellular signal-regulated kinase (ERK) and cAMP response element binding (CREB) in the hippocampus of scopolamine-induced mice. Taken together, our results indicate that LQ may be useful for the treatment of learning and memory impairments, and that the beneficial effects of LQ are mediated, in part, by cholinergic and BDNF/ERK/CREB signaling enhancement and/or protection.

Lonicera japonica THUNB. Extract Inhibits Lipopolysaccharide-Stimulated Inflammatory Responses by Suppressing NF-κB Signaling in BV-2 Microglial Cells.

In the current study, we evaluated the anti-inflammatory effects of Lonicera japonica THUNB. (LJ) and its underlying molecular mechanism in lipopolysaccharide (LPS)-stimulated BV-2 microglial cells. Our results indicated that LJ significantly inhibits LPS-stimulated production of nitric oxide (NO) and prostaglandin E2 (PGE2). In addition, LJ inhibited inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) at both the protein and mRNA levels. In LPS-stimulated BV-2 microglial cells, LJ inhibited proinflammatory cytokines and chemokines, tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), monocyte chemoattractant protein-1 (MCP-1), matrix metalloproteinase-9 (MMP-9) enzymatic activities, and/or mRNA expression, as well as reactive oxygen species (ROS) production. LJ significantly suppressed activation of nuclear factor-κB (NF-κB) and its translocation from the cytosol to the nucleus and suppressed the DNA-binding activity of NF-κB. Furthermore, LJ significantly inhibited phosphorylation of c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase 1/2 (ERK 1/2), p38 mitogen-activated protein kinases (MAPKs), phosphatidylinositol 3-kinases (PI3K)/Akt, and Janus kinase 1 (JAK1)/signal transducer and activator of transcription (STAT)1/3. Collectively, our findings indicated that the antineuroinflammatory properties of LJ in LPS-induced BV-2 microglial cells is due to downregulation of proinflammatory cytokines and chemokines downstream of inhibition of NF-κB activation.

Eucommia ulmoides Oliv. bark. attenuates 6-hydroxydopamine-induced neuronal cell death through inhibition of oxidative stress in SH-SY5Y cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Eucommia ulmoides Oliv. Bark. (EUE) has commonly been used to fortify the muscles and lungs, lower blood pressure, prevent miscarriage, improve liver and kidney tone, and promote longevity as a traditional tonic medicine in Korea, China, and Japan. AIM OF THE STUDY: In this study, we investigated the mechanisms by which EUE protects neuronal cells from apoptosis induced by the Parkinson's disease (PD)-related neurotoxin, 6-hydroxydopamine (6-OHDA). MATERIALS AND METHODS: We determined the neuroprotective effects of EUE on 6-OHDA-induced neuronal cell death, cytotoxicity, reactive oxygen species (ROS) production, and mitochondrial membrane dysfunction. Moreover, we examined whether EUE suppressed phosphorylation of c-Jun N-terminal kinase (JNK), phosphatidylinositol 3-kinase (PI3K)/Akt, and glycogen synthase kinase-3 beta (GSK-3ß). Furthermore, the neuroprotective effects of EUE on 6-OHDA-induced activation of nuclear factor-kappa B (NF-κB) was studied in SH-SY5Y cells. RESULTS: Pretreatment of SH-SY5Y cells with EUE significantly reduced 6-OHDA-induced cell death and cytotoxicity. EUE inhibited 6-OHDA-induced generation of ROS, which conferred cytoprotection against 6-OHDA-induced oxidative injury. EUE treatment also strikingly inhibited 6-OHDA-induced mitochondrial dysfunction. In addition, EUE suppressed phosphorylation of JNK, PI3K/Akt, and GSK-3ß. Furthermore, EUE blocked 6-OHDA-induced NF-κB nuclear translocation, an event downstream from JNK, PI3K/Akt, and GSK-3ß phosphorylation. Moreover, chlorogenic acid (CGA), one of the active constituents of EUE, was also able to reduce 6-OHDA-induced toxicity in SH-SY5Y cells. CONCLUSION: Taken together, these results suggest that EUE attenuates oxidative stress through activation of JNK, PI3K/Akt, GSK-3ß, and NF-κB pathways, thereby protecting cells from neuronal cell death.

Anxiolytic effects of Julibroside C1 isolated from Albizzia julibrissin in mice.

Julibroside C1 is a saponin-containing compound isolated from Albizzia julibrissin Durazz. In this study, we investigated the putative anxiolytic effects of Julibroside C1 using the elevated plus maze (EPM) in mice. Julibroside C1 at doses of 0.5 and 1 mg/kg significantly increased the time spent in the open arms and the number of entries into the open arms of the EPM compared to the control group. Moreover, the anxiolytic-like effects of Julibroside C1 (0.5 mg/kg) were blocked by WAY-100635 (5-HT1A receptor antagonist), bicuculline (GABA(A) receptor antagonist), and flumazenil (antagonist of the GABA(A) receptor benzodiazepine site). However, Julibroside C1 did not change locomotor activity or induce myorelaxant effects. We used quantitative receptor autoradiography to investigate the effects of Julibroside C1 on alterations in mouse brain receptors. After acute treatment with Julibroside C1 (0.5 mg/kg), [(3)H]-8-OH-DPAT binding was significantly decreased in the CA1 region of the hippocampus and [(3)H]-flunitrazepam binding was decreased remarkably in the cingulate cortex region. However, [(3)H]-muscimol binding did not show a significant change in any brain region. Taken together, our findings suggest that Julibroside C1 shows anxiolytic-like effects, which might be mediated by the 5-HT1A and GABA(A)-benzodiazepine receptor systems.

5-HT(1A) receptor binding in the dorsal raphe nucleus is implicated in the anxiolytic-like effects of Cinnamomum cassia.

Previously we reported that the 50% EtOH extract of Cinnamomum cassia (C. cassia) possesses anxiolytic-like activity in the mouse elevated plus maze (EPM) test. This activity was blocked by the 5-HT(1A) receptor antagonist, WAY 100635. Therefore, in order to investigate the effect of C. cassia on 5-HT(1A) receptor binding, quantitative autoradiography of 5-HT(1A) receptors was carried out in brains of mice treated acutely and repeatedly with C. cassia. Binding of [(3)H]8-OH-DPAT to the 5-HT(1A) receptor was investigated in the mouse brain. After a single treatment of C. cassia (750 mg/kg, p.o.), [(3)H]8-OH-DPAT binding showed a significant increase in the dorsal raphe nucleus (DRN). After repeated treatment with C. cassia (100mg/kg, once a day for 5 days, p.o.), [(3)H]8-OH-DPAT binding showed no significant change in any brain region. Taken together, the anxiolytic-like effect of the 50% EtOH extract of C. cassia might be mediated by region specific change of 5-HT(1A) receptors in the dorsal raphe nucleus.

Eucommia ulmoides Oliv. Bark. protects against hydrogen peroxide-induced neuronal cell death in SH-SY5Y cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Eucommia ulmoides Oliv. Bark. (EUE), has commonly been used to fortify the muscles and lungs, lower blood pressure, prevent miscarriage, improve the tone of liver and kidneys, and promote longevity the traditional tonic medicines of Korea, China, and Japan. AIM OF THE STUDY: In this study, we investigated that the neuroprotective activities and possible mechanisms of EUE aqueous extract in hydrogen peroxide (H(2)O(2))-induced neuronal cell death in human SH-SY5Y neuroblastoma cells. MATERIAL AND METHOD: We examined the effects of EUE against H(2)O(2)-induced cytotoxicity, DNA condensation, the production of reactive oxygen species (ROS), loss of mitochondria membrane potential (MMP), the proteolysis of cleaved poly-ADP-ribose polymerase (PARP), and the expression of Bcl-2, Bcl-xL, cleaved caspase-3, and release of cytochrome c. Moreover, we attempted to determine whether EUE suppressed the phosphorylation of c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase 1/2 (ERK 1/2), and phosphoinositide 3-kinase (PI3K)/Akt. RESULTS: Pretreatment with EUE increased cell viability and inhibited cytotoxicity and DNA condensation. EUE also attenuated the increase in ROS production and MMP reduction. Western blot data revealed that EUE inhibited H(2)O(2)-induced up- or down-regulation of cleaved PARP, cleaved caspase-3, Bcl-2, and Bcl-xL. The EUE inhibited release of cytochrome c from mitochondria to the cytosol, and significantly attenuated H(2)O(2)-induced phosphorylation of JNK, p38 MAPK, ERK 1/2, and PI3K/Akt. CONCLUSION: The potent neuroprotective capacity of EUE, shown in these experiments, may potentially be applied in the prevention or treatment of neurodegenerative diseases such as Alzheimer's disease (AD).

Gintonin, a ginseng-derived lysophosphatidic acid receptor ligand, attenuates Alzheimer's disease-related neuropathies: involvement of non-amyloidogenic processing.

Ginseng extracts show cognition-enhancing effects in Alzheimer's disease (AD) patients. However, little is known about the active components and molecular mechanisms of how ginseng exerts its effects. Recently, we isolated a novel lysophosphatidic acid (LPA) receptor-activating ligand from ginseng, gintonin. AD is caused by amyloid-ß protein (Aß) accumulation. Aß is derived from amyloid-ß protein precursors (AßPPs) through the amyloidogenic pathway. In contrast, non-amyloidogenic pathways produce beneficial, soluble AßPPα (sAßPPα). Here, we describe our investigations of the effect of gintonin on sAßPPα release, Aß formation, Swedish-AßPP transfection-mediated neurotoxicity in SH-SY5Y neuroblastoma cells, and Aß-induced neuropathy in mice. Gintonin promoted sAßPPα release in a concentration- and time-dependent manner. Gintonin action was also blocked by the Ca2+ chelator BAPTA, α-secretase inhibitor TAPI-2, and protein-trafficking inhibitor brefeldin. Gintonin decreased Aß1-42 release and attenuated Aß1-40-induced cytotoxicity in SH-SY5Y cells. Gintonin also rescued Aß1-40-induced cognitive dysfunction in mice. Moreover, in a transgenic mouse AD model, long-term oral administration of gintonin attenuated amyloid plaque deposition as well as short- and long-term memory impairment. In the present study, we demonstrated that gintonin mediated the promotion of non-amyloidogenic processing to stimulate sAßPPα release to restore brain function in mice with AD. Gintonin could be a useful agent for AD prevention or therapy.

Anxiolytic-Like Effects of Chrysanthemum indicum Aqueous Extract in Mice: Possible Involvement of GABAA Receptors and 5-HT1A Receptors.

Chrysanthemum indicum Linne is an ancient herbal medicine used to treat bone and muscle deterioration, ocular infl ammation, headache, and anxiety in Korea, China, and Japan. Furthermore, tea derived from Chrysanthemum indicum Linne has been used to treat anxiety by facilitating relaxation and curing insomnia. However, no reports exist on the anxiolytic-like effects of Chrysanthemum indicum Linne water extract (CWE) in mice. In the present study, we investigated the anxiolytic-like effects of CWE using the elevated plus-maze (EPM) test in mice. CWE, at a dose of 500 mg/kg (p.o.), signifi cantly increased the time spent in the open arms of the EPM compared to a vehicle-injected control group. Moreover, the effect of CWE (500 mg/kg) was blocked by bicuculline (a selective GABAA receptor antagonist) and WAY 100635 (a selective 5-HT1A receptor antagonist). Taken together, these fi ndings suggest that the anxiolytic-like effects of CWE might be mediated by the GABAA receptor and the 5-HT1A receptor.

The neuroprotective effects of Lonicera japonica THUNB. against hydrogen peroxide-induced apoptosis via phosphorylation of MAPKs and PI3K/Akt in SH-SY5Y cells.

We investigated the neuroprotective effects of Lonicera japonica THUNB. (Caprifoliaceae) (LJ) extract against hydrogen peroxide (H(2)O(2)), a toxin created by oxidative stress and implicated in neurodegenerative diseases, in human SH-SY5Y neuroblastoma cells. We examined the effects of LJ against H(2)O(2)-induced cytotoxicity, apoptosis, the production of reactive oxygen species (ROS), the proteolysis of cleaved poly-ADP-ribose polymerase (PARP), and the expression of Bcl-2, Bcl-xL, and cleaved caspase-3. Moreover, we attempted to determine whether LJ suppressed the phosphorylation of Akt, c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (MAPK), and extracellular signal-regulated kinase 1/2 (ERK 1/2). We found that LJ improved cell viability, inhibited cytotoxicity and apoptosis, and attenuated elevations in ROS and nuclear condensation. In addition, LJ showed radical scavenging ability in 2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2-azinobis-(3-ethyl-benzthiazoline-6-sulfonic acid) (ABTS) assays. Western blot data revealed that LJ inhibited H(2)O(2)-induced up- and down-regulation of cleaved PARP, cleaved caspase-3, Bcl-2, and Bcl-xL. Furthermore, LJ significantly attenuated the H(2)O(2)-induced phosphorylation of Akt, JNK, p38 MAPK, and ERK1/2. These results demonstrate that LJ possesses potent neuroprotective activity. Its potential to treat neurodegenerative diseases warrants further research.

Neuroprotective effects of Eucommia ulmoides Oliv. Bark on amyloid beta(25-35)-induced learning and memory impairments in mice.

In the present study, we examined whether aqueous extract of Eucommia ulmoides Oliv. Bark (EUE) with graded doses exerted its neuroprotective effects on amyloid beta(25-35) (Aß(25-35))-induced learning and memory impairments in mice. Mice received a single intracerebroventricular (i.c.v.) injection of Aß(25-35) 6 nmol as the critical factor in Alzheimer's disease (AD), cognition was evaluated using Y-maze, passive avoidance, and Morris water maze tests. EUE significantly improved the Aß(25-35)-induced memory deficit in the Y-maze test. Also, EUE increased step-through latency time with Aß(25-35)-induced learning and memory deficits in the passive avoidance test. In addition, EUE decreased the escape latencies with Aß(25-35)-induced cognitive impairments in the Morris water maze test. In the probe trial session, EUE increased time spent in the target quadrant. In the in vitro study, EUE was found to inhibit acetylcholinesterase (AChE) activity in a dose-dependent manner (IC50 value; 172 µg/ml). Ex vivo study, EUE significantly inhibited AChE activity in the hippocampus and frontal cortex. These results demonstrate that EUE possesses potent neuroprotective effects and that its beneficial effects are mediated, in part, by AChE inhibition, and therefore, might be a potential candidate in neurodegenerative diseases such as AD.

Neuroprotective effects of chlorogenic acid on scopolamine-induced amnesia via anti-acetylcholinesterase and anti-oxidative activities in mice.

Chlorogenic acid is a major polyphenolic component of many plants and beverages, and is particularly abundant in coffee. We evaluated the neuroprotective effects of chlorogenic acid on learning and memory impairment induced by scopolamine (0.5 mg/kg, i.p.), a muscarinic antagonist, using the Y-maze, passive avoidance, and Morris water maze tests. The chlorogenic acid significantly improved the impairment of short-term or working memory induced by scopolamine in the Y-maze test, and significantly reversed cognitive impairments in mice as measured by the passive avoidance test. In addition, chlorogenic acid decreased escape latencies in the Morris water maze test. In a probe trial session, chlorogenic acid increased the latency time in the target quadrant in a dose-dependent manner. Ex vivo, chlorogenic acid inhibited acetylcholinesterase activity in the hippocampus and frontal cortex. Chlorogenic acid also decreased malondialdehyde levels in the hippocampus and frontal cortex. In vitro, chlorogenic acid was found to inhibit acetylcholinesterase activity (IC50=98.17 µg/ml) and free radical scavenging activity (IC50=3.09 µg/ml) in a dose-dependent manner. These results indicate that chlorogenic acid may exert anti-amnesic activity via inhibition of acetylcholinesterase and malondialdehyde in the hippocampus and frontal cortex.