Effects Of Fructus Akebiae Extracts On The Level Of Monoamine Neurotransmitter In Rat Brain

Objective(1) To establish an accurate, sensitive, and simple method of high performance liquid chromatography-electrochemical detector (HPLC-ECD) for the determination of monoamine neurotransmitter in rat brain tissues;(2) To investigate the effects of Fructus Akebiae extracts (FAE) on the level of NA, DA and5-HT in the frontal cortex, hippocampus and striatum of rat, which provided a better understanding on the antidepressant mechanism of FAE;(3) To establish a high performance liquid chromatography-diode array detector (HPLC-DAD) method for the determination of Aristolochic Acid A in FAE and Hederagenin, which improved the quality standard and ensured the security of FAE and Hederagenin.Methods1. The developed method for the monoamine neurotransmitter determination in rat brain tissues:The HPLC-ECD method was applied to the quantitative determination of NA, DA and5-HT in the brain tissues of rat with the DHBA as the internal standard. Separation was carried out on the Dikma Diamonsil-C18chromatographic column (5μm,4.6×250mm) at a flow rate of1.0mL/min and the column temperature was32℃. The mobile phase was NaH2PO4solution-acetonitrile (90:10), in which the NaH2PO4solution contained25.0mmol/L NaH2PO4,1.7mmol/L OSA,0.7mmol/L triethylamine, and0.025mmol/L EDTA-2Na (pH=3.0). The detection voltages of ECD were-150V (E1) and220V (E2), and the injection volume was20μL.2. Methods involved in the study of the effects of FAE on the level of monoamine neurotransmitter in rat brain:(1) For the acute administration, the male SD rats,200～220g, were randomly divided into4groups:vehicle group (0.5%CMC-Na solution),25mg/kg FAE group,50mg/kg FAE group and10mg/kg Escitalopram (ESC) group. All the drugs were given via the oral route with the dose of10mL/kg. Brain tissues were collected on the ice at each time-point of1h,2h,3h, and5h after administration, respectively.(2) For the subacute administration, the male SD rats,200～220g, were randomly divided into4groups as mentioned above. All the drugs were given via the oral route for21d with the dose of10mL/kg, and brain tissues were collected at2h after the last administration.(3) Sample preparation:All the animals were killed by decapitation and their brains were rapidly harvested. Then frontal cortex, hippocampus and striatum were separated on the ice and stored in liquid nitrogen. The collected tissues were homogenized with perchloric acid working solution for2min under the ice-bath after weighted. Supernatant fluid was obtained after centrifugation (I,4000×g,20min,4℃) and then filtrated with the0.22μm membrane before the HPLC-ECD determination.(4) Data processing:All the data were transformed into wet weight of tissue (ng/g). Results were analyzed statistically with ONE-WAY ANOVA by SPSS13.0and plotted through GraphPad Prism5.3. The developed method for Aristolochic acid A determination:The HPLC-DAD method was applied to the quantitative determination of Aristolochic acid A determination. Samples were separated via C18chromatographic column (5μm,4.6×250mm), and the column temperature was maintained at32℃. The mobile phase was acetonitrile-water-glacial acetic acid (80:20:0.04). The flow rate was1.0mL/min and the injection volume was20μL. The wavelengths of determination were210nm and250nm.Results1. The HPLC-ECD method for the monoamine neurotransmitter determination in rat brain tissues: All target compounds were separated well without the disturbance of endogenous substance in biological samples using the established HPLC-ECD method. The standard curves of NA, DA and5-HT were linear (R2>0.9996) with the lower limits of quantification (LLOQ):12.0ng/mL,9.8ng/mL and20.8ng/mL respectively. The recovery of NA, DA and5-HT ranged from83.74%to104.61%. The intra day precisions were in the range of3.05%-5.81%, and the inter day precisions were between4.98%and7.59%, which showed good reproducibility for all target compounds (less than15%).2. The acute effect on the level of monoamine neurotransmitter in rat brain:(1) Compared with the vehicle group, the level of NA in frontal cortex and hippocampus at3h after administration increased significantly in25mg/kg FAE group and50mg/kg FAE group, while the level of5-HT increased in10mg/kg ESC group at2h after administration and in50mg/kg FAE group at3h after administration. However, none significant effects on DA levels were observed in all groups.(NA-vehicle group vs the25mg/kg and the50mg/kg FAE groups, in frontal cortex:360.645±13.619ng/g vs436.582±5.785ng/g,450.396±12.506ng/g; in hippocampus:351.405±14.206ng/g vs394.362±8.041ng/g,410.530±10.477ng/g.)(5-HT-vehicle group vs10mg/kg ESC group, in frontal cortex:934.494±25.054ng/g vs1034.001±29.773ng/g; in hippocampus:592.150±11.557ng/g vs645.036±12.545ng/g. Vehicle group vs50mg/kg FAE group, in frontal cortex:939.202±27.075ng/g vs1024.500±39.169ng/g; in hippocampus:593.729±22.887ng/g vs645.012±3.969ng/g.)(2) Compared with vehicle group, the DA level in striatum at3h after administration increased in25mg/kg FAE group and50mg/kg FAE group, while levels of NA and5-HT didn’t show significant variation in all groups.(DA-vehicle group vs the25mg/kg and the50mg/kg FAE groups,7316.341±463.518ng/g vs9057.434±604.403ng/g,9205.288±832.326ng/g.)3. The subacute effect on the level of monoamine neurotransmitter in rat brain:(1) Compared with the vehicle group, the level of NA in frontal cortex and hippocampus increased significantly in FAE (25mg/kg and50mg/kg) groups, and the level of5-HT in frontal cortex and hippocampus increased significantly in FAE (25mg/kg and50mg/kg) groups and10mg/kg ESC group after administration for21d. However, none significant effects on DA levels were observed in all groups.(NA-vehicle group vs the25mg/kg and the50mg/kg FAE groups, in frontal cortex:302.295±7.036ng/g vs351.930±3.271ng/g,382.463±7.208ng/g; in hippocampus:279.287±4.142ng/g vs302.511±6.426ng/g,394.189±4.307ng/g.)(5-HT-vehicle group vs the10mg/kg ESC group, the25mg/kg FAE group and the50mg/kg FAE group, in frontal cortex:826.005±9.528ng/g vs1128.418±17.316ng/g,1022.831±19.777ng/g,1012.098±15.274ng/g; in hippocampus:549.010±2.723ng/g vs780.082±4.558ng/g,703.941±3.976ng/g,758.575±2.915ng/g.)(2) Compared with vehicle group, the DA level in striatum increased significantly in25mg/kg FAE group and50mg/kg FAE group after administration for21d, while levels of NA and5-HT didn’t show significant variation in all groups.(DA-vehicle group vs the25mg/kg and the50mg/kg FAE groups,7029.182±492.037ng/g vs9500.078±424.739ng/g,9706.304±366.826ng/g.)4. The HPLC-DAD method for Aristolochic acid A determination:Aristolochic acid A was well-separated from Hederagenin with HPLC method. The standard curve was linear in the range of0.24～2.4μg/mL (R2=0.9993), and the limits of detection (LOD) was40ng/mL. The recovery ranged from85.96%to96.23%. The intra day precision was in the range of1.24%～5.28%, and the inter day precision was between4.11%and7.28%. No Aristolochic acid A was detected in FAE and Hederagenin.ConclusionAn accurate, sensitive, and simple HPLC-ECD method has been successfully developed and applied to the determination of monoamine neurotransmitter in the brain tissues of rat for the acute and subacute administration. The results suggest that the antidepressant mechanism of FAE is relevant to the elevation of monoamine neurotransmitter. The rapid and simple HPLC-DAD method has been utilized for the determination of Aristolochic acid A, and the results reveal that the FAE and Hederagenin in this experiment is safe.