| Objective:(1)The objective of our present research work was to survey the neuroprotective effect of the ethanol extract of Scutellaria baicalensis Georgi(S.B)on the excitotoxic neuronal cell death in primary rat cortical cell cultures.(2)Phytochemical analysis of the bioactive extract was carried out by a combination of HPLC and LC-ESI-MS analytical techniques.Methods:(1)Ethanol(95%)was used for hot extraction which was carried out for 4 hours using a soxhlet extraction apparatus,and the extract was then concentrated under reduced pressure in a rotary evaporator at 40 °C.(2)The inhibitory effects of the S.B extract on the neuronal cell death were qualitatively and quantitatively estimated by phase-contrast microscopy and lactate dehydrogenase(LDH)assays.(3)Using radioligand([3H]MDL105,519 and[3H]MK-801)binding assayes,to observe that the inhibitory effect of the extract was more potent and discriminatory for the N-methyl-D-aspartate(NMDA)receptor mediated toxicity.(4)Liquid chromatograp-hy-tandem mass spectrometry(LC-ESI-MS-MS)/HPLC analysis were used to detect the active constituents of the extract.Results:(1)We observed that primary cortical neurons in normal control group exhibited a characteristic of neurons by phase contrast microscopy,and the axons and dendrites grew well and connected into a network.However,Glu and NMDA induced a characteristic of neurodegeneration,including acute neuronal swelling,breakage in dendrocytes and the indistinct nuclear shape.However,S.B extract-treated neurons exhibited features with clear axons and dendrites,which indicated that S.B extraxt could inhibit the excitotoxic induced-neuronal damage.(2)The degree of neuronal cell death was quantitatively estimated by calculating LDH release activities given off by the damaged neurons out into the culture media.Our results demonstrated that when the neuronal cell cultures were exposed for 20 min to Glu(350 μM)in the presence of different concentrations of the S.B extract,the Glu-induced excitotoxicity was dramatically inhibited in a dose-dependent pattern.At the concentration of 100 pg/ml,almost 90-95%of the neurons were secured from the excitotoxic insults.The IC50 value of the extract was found to be 60.01 and 28.60 Vg/ml respectively for Glu and NMDA induced excitotoxicity respectively.(3)It was observed that at a concentration of 100 μg/ml of the extract,more than 90%of the binding of[3H]MDL105,519 was displaced by the extract.The IC50 value was found to be 35.1 μg/ml.In addition,at a concentration of 100 μg/ml,[3H]MK-801 binding with the receptor site was around 20%of the control binding alculated in the absence of the extract.The IC50 value of the extract was found to be 65.1 μg/ml.(4)The phytochemical analysis of the S.B ethanol extract was carried out by LC-ESI-MS in combination with HPLC-DAD analyticaltechniques.The six chemical constituents identified were wogonin,oroxylin A,Baicalein,wogonoside,Scutellarin,Baicalin.The amount of isolated constituents present in Scutellaria baicalensis was to be 0.316%(Wogonin),0.105%(Oroxylin A),0.182%(Baicalein),0.112%(wogonoside),0.092%(Scutellarin)and 0.089%(Baicalin)per mg of the extract.Conclusion:(1)The S.B ethanol extract exhibited a potent and dose dependent inhibition of the Glu-induced excitotoxicity in the culture media.Furthermore,the S.B ethanol extract exhibited neuroprotection against excitotoxic cell death and this neuroprotection was principally mediated through the inhibition of NMDA receptor function by interacting with the glycine binding site of the NMDA receptor.(2)Phytochemical analysis of the bioactive extract revealed the presence of six phytochemical constituents including baicalein,baicalin,wogonin,wogonoside,scutellarin and Oroxylin A. |
