• Archives of Pharmacal Research
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• v.29 no.8
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• pp.699-706
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• 2006

The present study evaluated antioxidant and neuroprotective activities of hesperidin, a flavanone mainly isolated from citrus fruits, and its aglycone hesperetin using cell-free bioassay system and primary cultured rat cortical cells. Both hesperidin and hesperetin exhibited similar patterns of 1,1-diphenyl-2-picrylhydrazyl radical scavenging activities. While hesperidin was inactive, hesperetin was found to be a potent antioxidant, inhibiting lipid peroxidation initiated in rat brain homogenates by $Fe^{2+}$ and L-ascorbic acid. In consistence with these findings, hesperetin protected primary cultured cortical cells against the oxidative neuronal damage induced by $H_2O_2$ or xanthine and xanthine oxidase. In addition, it was shown to attenuate the excitotoxic neuronal damage induced by excess glutamate in the cortical cultures. When the excitotoxicity was induced by the glutamate receptor subtype-selective ligands, only the N-methyl-D-aspartic acid-induced toxicity was selectively and markedly inhibited by hesperetin. Furthermore, hesperetin protected cultured cells against the $A_{{\beta}(25-35)}-induced$ neuronal damage. Hesperidin, however, exerted minimal or no protective effects on the neuronal damage tested in this study. Taken together, these results demonstrate potent antioxidant and neuroprotective effects of hesperetin, implying its potential role in protecting neurons against various types of insults associated with many neurodegenerative diseases.

• Journal of Life Science
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• v.15 no.4 s.71
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• pp.600-606
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• 2005

Neurofilament (NF) proteins constitute the major intermediate filament type in adult neurons. They are made up by the copolymerization of the neurofilament light (NF-L, 61 kDa), medium (NF-M, 90kDa), and heavy (NF-H, 115 kDa) proteins. Although neurofilaments play a crucial .ole in neuronal growth, organization, shape, and plasticity, their expression pattern and cellular distribution in the developing neurons remain unknown. In this study, we have produced a rabbit polyclonal antibody specific to NF-M and investigated expression of NF-M in cultured cortical neurons. Immunostaining of 12 and 24 h cultures revealed strong expression of NF-M in axonal growth cone and in the region of a soma toward the axon. Doublestaining of 4 and 14 DIV corical neurons with NF-M and PSD95 antibodies revealed that both axon and dendrites were stained intensely with NF-M antibody, and that NF-M immunostaining along dendrites is often punctate and colocalize with PSD95 puncta, indicating that the puncta represent postsynaptic spines. Presence of NF-M in the postsynaptic spine was also indicated by immunoblot analysis of the postsynaptic density fraction. Taken together, our results show intensive targeting of NF-M into axons in the early axonal development, and into spines in mature neurons, indicating its important functions in axon and spine development.

• Korean Journal of Medicinal Crop Science
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• v.13 no.5
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• pp.219-226
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• 2005

Sanguisorbae radix (SR) from Sanguisorba officinalis L. (Losaceae) is widely used in Korea and China due to its various pharmacological activity. The present study aims to investigate the effect of the methanol extract of SR on amyloid ${\beta}$ Protein(25-35) $(A{\beta}\;(25-35))$, a synthetic 25-35 amyloid peptide, -induced neurotoxicity using cultured rat cortical neurons. SR, over a concentration range of $10-50\;{\mu}g/ml$, inhibited the $A{\beta}$ (25-35) $(10\;{\mu}M)-induced$ neuronal cell death, as assessed by a 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assay and the number of apoptotic nuclei, evidenced by Hoechst 33342 staining. Pretreatment of SR $(50\;{\mu}g/ml)$ inhibited $10\;{\mu}M\;A{\beta}$ (25-35)-induced} elevation of cytosolic calcium concentration $([Ca^{2+}]c)$, which was measured by a fluorescent dye, fluo-4 AM. SR $(10\;and\;50\;{\mu}g/ml)$ inhibited glutamate release into medium induced by $10\;{\mu}M\;A{\beta}(25-35)$, which was measured by HPLC, and generation of reactive oxygen species. These results suggest that SR prevents $A{\beta}$ (25-35)-induced neuronal cell damage in vitro.

• Journal of Life Science
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• v.19 no.6
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• pp.756-764
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• 2009

Scutellariae baicalensis GEORGt Acorus gramineus SOLAND and Gastrodia elata BLUME are traditional medicines used in the treatment of incipient stoke. In this study we investigated their effects on various aspects of neuronal differentiation in single or composite forms. Water-extracts of these medicines showed neuroprotective effects on cultured rat cortical neurons in normoxia and hypoxia. To understand the mechanism for neuroprotection we carried out various cell biological assays. They stimulated initial differentiation of neuronal development (transition from stage 1 to 2), and increased the number of spines and the length and number of dendritic processes. These effects were best manifested in the experimental group, which were given a mixture of the three kinds of extracts (p<0.01). To assess improvement of brain functions we carried out Morris water-maze tests for the mice that were fed on these extracts instead of water for 4 weeks. The experimental groups, especially those which were given the mixture of the three kinds of extract, showed significant (p<0.01) enhancement in memory as early as one day after the learning trial. These results indicate that these three kinds of extracts have synergistic effects on neuronal protection and improvement of brain functions.

• The Journal of Korean Medicine
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• v.24 no.3
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• pp.72-83
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• 2003

Objective : The goal of the present study was to investigate the protective effect of Gamiheechum-tang (Jiaweixiqian-tang; GHCT) on brain tissue damage from chemical or ischemic insults. Methods : Levels of cultured cortical neuron death caused by toxic chemicals were measured by LDH release assay. Neuroprotective effects of GHCT on brain tissues were examined in vivo by ischemic model of middle cerebral artery (MCA) occlusion. Results : Animal groups treated with GBCT showed significantly decreased hypertension, and reduced levels of aldosterone, dopamine, and epinephrine in the plasma. GHCT treatments ($l0-200\mu\textrm{g}/ml$) significantly decreased cultured cortical neuron death mediated by AMPA, kainate, BSO, or Fe2+ when measured by LDH release assay. Yet, cell death mediated by NMDA was effectively protected by GHCT at the highest concentration examined ($200\mu\textrm{g}/ml$). In the in vivo experiment examining brain damage by MCA occlusion, affected brain areas by ischemic damage and edema were significantly less in animal groups administered with GHCT compared to the non-treated control group. Neurological examinations of forelimbs and hindlimbs showed that GHCT treatment improved animals' recovery from ischemic injury. Moreover, the extent of injury in cortical and hippocampal pyramidal neurons in ischemic rats was much reduced by GHCT, whose morphological features were similarly observed in non-ischemic animals. Conclusion : The present data suggest that GBCT may play an important role in protecting brain tissues from chemical or ischemic injuries.

• Korean Journal of Medicinal Crop Science
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• v.13 no.2
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• pp.95-102
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• 2005

Caulis Bambusae in Taenia is widely used in Korea and China due to its various pharmacological activity. The present study aims to investigate the effect of the methanol extract of Caulis Bambusae in Taenia (CB) from Phyllostachys nigra Munro var. henonis Stapf (Gramineae) on amyloid ${\beta}$ protein (25-35) $(A{\beta}\;(25-35))$, a synthetic 25-35 amyloid peptide, -induced neurotoxicity using cultured rat cortical neurons. CB, over a concentration range of $10-50{\mu}g/{\mu}l$, inhibited the $A{\beta}\;(25-35)\;(10\;{\mu}M)$-induced neuronal cell death, as assessed by a 3-[4,5-dimethyIthiazole-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assay and the number of apoptotic nuclei, evidenced by Hoechst 33342 staining. CB $(50\;{\mu}g/{\mu}l)$ inhibited glutamate release into medium induced by $10\;{\mu}M\;A{\beta}$, (25-35) which was measured by HPLC. Pretreatment of CB $(50\;{\mu}g/{\mu}l)$ inhibited $10{\mu}M\;A{\beta}$ (25-35)-induced elevation of cytosolic calcium concentration $([Ca^{2+}]_c)$, which was measured by a fluorescent dye, fluo-4 AM, and generation of reactive oxygen species. These results suggest that CB prevents $A{\beta}$ (25-35)-induced neuronal ell damage in vitro.

• Journal of Sasang Constitutional Medicine
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• v.11 no.2
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• pp.267-281
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• 1999

1. Purpose : The purpose of this study was to determine the effects of Taeyeumjoweetang on the cerebral neurons injured by glucose oxidase(GO). 2. Methods : I observed cell viability in mouse cerebral neurons exposed to glucose oxidase by NR assay and MTT assay and determined lipid peroxidation in mouse cerebral neurons exposed to glucose oxidase. After administration of Taeyeumjoweetang water extracts, I observed significant changes of cell viability, lipid peroxidation in mouse cerebral neurons exposed to glucose oxidase. 3. Results : GO induced cell degeneration such as the decrease of cell viability was measured by MTT and NR assay in the cultured mouse cerebral cortical neurons. Taeyeumjoweetang was effective in the increase of total protein of neurons inhibited by GO. Taeyeumjoweetang was effective in the decrease of lipid peroxidation of neurons produced by GO.

• YAKHAK HOEJI
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• v.46 no.3
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• pp.185-191
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• 2002

Alzheimer's disease (AD) involves neuronal degeneration with impaired cholinergic transmission, particularly in areas of the brain associated with learning and memory. Several cholinesterase inhibitors are widely prescribed to ameliorate the cognitive deficits in AD patients. In an attempt to examine if tacrine and donepezil, two well-known cholinesterase inhibitors, exhibit additional pharmacological actions in primary cultured rat cortical cells, we investigated the effects on neuronal injuries induced by glutamate or N-methyl-D-aspartate (NMDA), $\beta$-amyloid fragment ( $A_{{beta}25-35)}$), and various oxidative insults. Both tacrine and donepezil did not significantly inhibit the excitotoxic neuronal damage induced by glutamate. However, tacrine inhibited the toxicity induced by NMDA in a concentration-dependent fashion. In addition, tacrine significantly inhibited the $A_{{beta}25-35)}$-induced neuronal injury at the concentration of 50 $\mu$M. In contrast, donepezil did not reduce the NMDA- nor $A_{{beta}25-35)}$-induced neuronal injury. Tacrine and donepezil had no effects on oxidative neuronal injuries in cultures nor on lipid peroxidation in vitro. These results suggest that, in addition to its anticholinesterase activity, the neuroprotective effects by tacrine against the NMDA- and $A_{{beta}25-35)$-induced toxicity may be beneficial for the treatment of AD. In contrast, the potent and selective inhibition of central acetylcholinesterase appears to be the major action mechanism of donepezil.

• Natural Product Sciences
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• v.21 no.2
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• pp.134-140
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• 2015

The present study aims to investigate the effect of methanol extract of Korean mistletoe (KM; Viscum album var. coloratum), on amyloid $\beta$ protein ($A\beta$) (25-35), a synthetic 25-35 amyloid peptide, -induced neurotoxicity in cultured rat cerebral cortical neurons and memory impairment in mice. Exposure of cultured neurons to $10{\mu}M$ $A\beta$ (25-35) for 24 h induced a neuronal cell death, which was measured by a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assay and Hoechst 33342 staining. KM (10, 30 and $50{\mu}g/ml$) significantly inhibited the $A\beta$ (25-35)-induced apoptotic neuronal death. KM ($50{\mu}g/ml$) inhibited 10 μM Aβ (25-35)-induced elevation of intracellular calcium concentration ([Ca²⁺]_i), which was measured by a fluorescent dye, Fluo-4 AM. Glutamate release into medium and generation of reactive oxygen species (ROS) induced by $10{\mu}M$ $A\beta$ (25-35) were also inhibited by KM (10, 30 and $50{\mu}g/ml$). These results suggest that KM may mitigate the $A\beta$ (25-35)-induced neurotoxicity by interfering with the increase of [Ca²⁺]_i and then inhibiting glutamate release and generation of ROS in cultured neurons. In addition, orally administered KM (25 and 50 mg/kg, 7 days) significantly prevented memory impairment induced by intracerebroventricular injection of $A\beta$ (25-35) (8 nmol). Taken together, it is suggested that anti-dementia effect of KM is due to its neuroprotective effect against $A\beta$ (25-35)-induced neurotoxicity and that KM may have therapeutic role in prevention of the progression of Alzheimer's disease.

• YAKHAK HOEJI
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• v.49 no.1
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• pp.30-37
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• 2005

Xuesaitong Ruanjiaonang (XR), a soft capsule containing Panax notoginseng saponins as main ingredients, is believed to remove extravasated blood and increase cerebral blood flow by improving blood circulation, and therefore, has been used in China to treat ischemic stroke or hemiplegia caused by cerebral thrombosis. To characterize pharmacological actions of XR, the present study evaluated its effects on neuronal cell damage induced by various oxidative insults or excitotoxic amino acids in primary cultured rat cortical cells. The neuronal cell viability was not affected by XR with the exposure for 2 h at the concentrations tested in this study ($10{\sim}1000\;{\mu}g/ml$). However, significant reduction of the cell viability was observed when the cultured cells were exposed to XR at $1000\;{\mu}g/ml$ for 24 h. XR was found to concentration-dependently inhibit the oxidative neuronal damage induced by $H_{2}O_2$, xanthine/xanthine oxidase or $Fe^{2+}$/ascorbic acid. In addition, it dramatically inhibited the excitotoxic damage induced by glutamate or N-methyl-D-aspartate (NMDA). We found that the NMDA-induced neurotoxicity was inhibited more effectively and potently than the glutamate-induced toxicity. Moreover, XR was found to exert mild inhibition of lipid peroxidation induced by $Fe^{2+}$/ascorbic acid in rat brain homogenates and some 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity. Taken together, these results demonstrate neuroprotective and antioxidant effects of XR, showing inhibition of oxidative and excitotoxic damage in the cultured cortical neurons, as well as inhibition of lipid peroxidation and its radical scavenging activity. Considering that excitotoxicity and oxidative stress pl ay crucial roles in neuronal cell damage during ischemia and reperfusion, these results may provide pharmacological basis for its clinical usage to treat ischemic stroke.