ABSTRACT
A sensitive and efficient liquid chromatography-mass spectrometry (LC-MS) method was developed and validated for the simultaneous determination of geniposide, 6α-hydroxygeniposide, and genipin gentiobioside in rat plasma. After the addition of internal standard (I.S.) salidroside and acidification (formic acid, 0.1%), plasma samples were carried out by protein precipitation with acetonitrile and separated on a Kromasil C(18) column (200 mm × 4.6 mm, 5 µm) within a runtime of 15.0 min. The linear ranges were 2-250 ng/mL for both 6α-hydroxygeniposide and genipin gentiobioside and 2-2000 ng/mL for geniposide, respectively. The lower limit of quantification (LLOQ) was 2 ng/mL for all the analytes. The validated method was successfully applied to the pharmacokinetics study of geniposide, 6α-hydroxygeniposide, and genipin gentiobioside in rats after oral administration of Zhi-zi-chi decoction.
Subject(s)
Drugs, Chinese Herbal/administration & dosage , Gardenia/chemistry , Glycine max/chemistry , Iridoids/blood , Mass Spectrometry/methods , Administration, Oral , Animals , Chromatography, High Pressure Liquid/methods , Male , Rats , Rats, WistarABSTRACT
AIM: Tau hyperphosphorylation and amyloid ß-peptide overproduction, caused by altered localization or abnormal activation of glycogen synthase kinase-3ß (GSK-3ß), is a pathogenic mechanism in Alzheimer's disease (AD). Valproic acid (VPA) attenuates senile plaques and neuronal loss. Here, we confirmed that VPA treatment improved spatial memory in amyloid precursor protein (APP)/presenilin 1 (PS 1) double-transgenic mice and investigated the effect of VPA on synaptic structure and neurite outgrowth. METHODS: We used ultrastructural analysis, immunocytochemistry, immunofluorescence staining, and Western blot analysis to assess the effect of VPA treatment in mice. RESULTS: VPA treatment thickened the postsynaptic density, increased the number of presynaptic vesicles, and upregulated the expression of synaptic markers PSD-95 and GAP43. VPA increased neurite length of hippocampal neurons in vivo and in vitro. In VPA-treated AD mouse brain, inactivated GSK-3ß (pSer9-GSK-3ß) was markedly increased, while hyperphosphorylation of tau at Ser396 and Ser262 was decreased; total tau levels remained similar. VPA treatment notably improved pSer133-cAMP response element-binding protein (CREB) and brain-derived neurotrophic factor (BDNF) levels, which are associated with synaptic function and neurite outgrowth. CONCLUSION: VPA improves behavioral deficits in AD, modifies synaptic structure, and accelerates neurite outgrowth, by inhibiting the activity of GSK-3ß, decreasing hyperphosphorylated tau, enhancing CREB and BDNF expression.
Subject(s)
Alzheimer Disease/drug therapy , Alzheimer Disease/pathology , Enzyme Inhibitors/therapeutic use , Glycogen Synthase Kinase 3/metabolism , Neurites/drug effects , Signal Transduction/drug effects , Synapses/drug effects , Valproic Acid/therapeutic use , Alzheimer Disease/complications , Alzheimer Disease/genetics , Amyloid beta-Protein Precursor/genetics , Animals , Brain/pathology , Cells, Cultured , Enzyme Inhibitors/pharmacology , Glycogen Synthase Kinase 3 beta , Humans , Male , Memory Disorders/drug therapy , Memory Disorders/etiology , Mice , Mice, Transgenic , Nerve Tissue Proteins/metabolism , Neurites/pathology , Neurites/ultrastructure , Neurons/drug effects , Neurons/pathology , Presenilin-1/genetics , Signal Transduction/genetics , Synapses/pathology , Up-Regulation/drug effects , Up-Regulation/genetics , Valproic Acid/pharmacology , tau Proteins/metabolismABSTRACT
A sensitive and specific liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) method was developed and validated for the determination of PAC-1 in rat plasma. After extraction with ethyl acetate, the chromatographic separation was carried out on an ACQUITY UPLC™ BEH C(18) column, with acetonitrile and water (39:61 (v/v) both containing 0.1% formic acid) as mobile phase at a flow rate of 0.20 mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring (MRM) mode via electrospray ionization (ESI) source. The calibration curve was linear over the range of 10-1500 ng/mL (r>0.99). The LOQ was evaluated to be 0.3 ng/mL. The method described herein is sensitive, selective and faster than other existing method, and was successfully applied to the pharmacokinetic study and gender difference investigation of PAC-1 after oral administration in rats.