| Background and ObjectiveParkinson's disease (PD) is a central nervous system diseases, featured mainly by progressive neurodegeneration and reduce of DA content in the system of substantia nigral and striatum. Generally replacement therapy with L-DOPA, it conversion to DA, and abate the symptoms. But long-time administration can weaken L-DOPA curative effects and caused side effects. There a reduction of DA storage ability in neuron with the development of PD, when blood concentration of L-DOPA over the response threshold, and no buffer effect of neuron to DA fluctuation. L-DOPA oxidation and releases free radical, may improve process of PD. Thus rational administration of L-DOPA is important. Shouwu Fang is a clinical effective Chinese herbal formula, it consists of radix polygoni multiflori preparata, pilose Antler, Gastrodia elata, et al. It has been found that the treatment with integration of Shouwu Fang and western medicine can not only increase therapeutic action, but also reduce toxic and side-effects.Microdialysis is an in vivo sampling technique permitting the continuous determination of test substances. Recently, this technique has been used in pharmacokinetics-pharmacodynamics study to determine the relationship between drug's concentration, effects and time.The experiment combined dual blood-brain sites microdialysis technique with HPLC-FLD and HPLC-ED to synchronous monitor the level of L-DOPA in blood and striatum, and neurotransmitter in striatum. To observe the effects of Shouwu Fang on pharmacokinetics of Levodopa in blood and extracellular fluids of striatum, and the effects on neurotransmitter, such as monoamine transmitters and hydroxyl free radical, in normal and PD rats. And then, further analysis the data with method of pharmacokinetics-pharmacodynamics study, to investigate the mechanism of Shouwu Fang on effects of L-DOPA and treatment of PD.Methods1 The method of measuring the L-DOPA with HPLC-FLD. Aglient 1200 system was adopted with a fluorescence detector (λex=278nm,λem=325nm), a MERCK Lichrospher 100 C18 column (5μm,4mm×250mm), a Nova-Pak C18 pre-column, a mobile phase consisted of 90% solution (containing EDTA 0.08mmol·L-1, KH2PO4 70mmol·L-1, solium heptanesulfonate 2.08mmol·L-1) and 10% methanol, and using a flow rate of 1.0ml·min-1, temperature with 35℃.2 The method of synchronous measuring the neurotransmitter, hydroxyl free radical and L-DOPA with HPLC-ED. We used a Antec Leyden BV C18 column (3μm, 2.1mm×100mm), a Antec DecadeⅡSDC electrochemical detector was set at 0.52v with a glass carbon electrode and a Ag/AgCl reference electrode. The mobile phase (pH 3.32, adjusted with phosphoric acid)consisted of EDTA (0.027mmol·L-1), sodium octane sulfonate (0.74mmol·L-1), KCL (2mmol·L-1), KH2PO4(100mmol·L-1), methanol(15%), acetonitrile (1%), acetic acid (0.05%), using a flow rate of O.2mL·min-13 To establish the PD rats model by injecting 6-OHDA into striatum. In this study, seven days before sampling, rats were anesthetized with sodium pentobarbital (40mg·kg-1) and placed on a stereotaxic frame. Unilateral injection of 6-OHDA (32μg·kg-1 1μL·min-1)was performed in striatum (coordinates with respect to bregma:A:+0.2mm, L:+3mm, V:7.5mm). The same volume normal saline was injected into the striatum of the control group rats.4 Effects of Shouwu Fang on pharmacokinetics of L-DOPA in blood and extracellular fluids of striatum and neurotransmitter in normal and PD rats.Rats grouping and dosage. Rats were randomly divided into six groups:control group, model group, control high dose group(L-DOPA 48mg-kg-1 and benserazide 12mg·kg-1), model high dose group(L-DOPA 48mg-kg-1 and benserazide 12mg·kg-1), model low dose group(L-DOPA 24mg·kg-1 and benserazide 6mg-kg-1), SWF+model low dose (L-DOPA 24mg·kg-1 and benserazide 6mg·kg-1, and Shouwu Fang 18g·kg-1 equivalent to herbs). The model groups establish the PD rats model by method above. The drugs or water were ig to rats for 6 days.Operation and blood-brain microdialysis. The rats were anesthetized with same method on the 6th day, the blood microdialysis probe was positioned within the jugular vein toward the right atrium and then was perfused with compound sodium chloride solution at a rate of 2.5μL·min-1. A piece of tubing was looped subcutaneously on the back of the rats to the surface of the neck from the abdominal space for the administration of drugs.On the 7th day of the experiment, a probe was inserted into the striatal guide, in freely moving rats. The blood and brain microdialysis probes were perfused with compound sodium chloride solution at a flow rate of 2.5μL·min-1 for 60 min, then the brain microdialysis probe was perfused with compound sodium chloride solution containing 5mmol·L-1 salicylic acid sodium salt. After a 60-min stabilization period, L-DOPA and benserazide were ip to rats at same dosage above. Brain and blood dialysates were collected in 15 min intervals for 420 min. Result1 The method of measuring the concentration of L-DOPA in blood with HPLC-FLD. Intra-day and inter-day RSD for L-DOPA(0.3125-5mg·L-1) were≤4.9%. correlation coefficients of linear regression equations from 0.3125 mg·L-1 to 5mg·L-1was 0.9987. the minimum detection limit of L-DOPA was 19μg·L-2 The method of synchronous measuring the neurotransmitter, hydroxyl free radical and L-DOPA with HPLC-ED. The 10mix standard liquid were consisted of L-DOPA, DA, DOPAC,5-HIAA,5-HT,2,3-DHBA,2,5-DHBA, EP, NE and HVA. Intra-day and inter-day RSD for 5 different concentrations of 10mix were≤8.7%. Correlation coefficients of linear regression equations of the each substance in 10 mix were≥0.9991。3 Synchronous pharmacokinetics study on Levodopa in PD rats blood and extracellular fluids of striatum. Both in normal and PD rats, drugs were absorbed in blood rapidly after administration through intraperitoneal injection, then entered the striatum through blood brain barrier. Both the blood and striatum concentration-time profiles of L-DOPA were described by a one compartment model. Tmax, AUC(o-∞) and Cmax in striatum were significantly different from that in blood.4 The relativity between the concentration of L-DOPA in blood, concentration of L-DOPA in target organ, and effects in target organ in PD rats. The target of pharmacokinetics is concentrations of L-DOPA in rat's blood or extracellular fluids of striatum. The target of pharmacodynamics is the level of DA in extracellular fluids of striatum, witch expressed asΔDA((determination value -basic value)/basic value). After administration of L-DOPA, ip, the L-DOPA concentration andΔDA value in extracellular fluids of striatum were increased, with the blood L-DOPA concentration was increased. The RSD showed strong correlation between L-DOPA concentration in blood and in extracellular fluids of striatum, between L-DOPA concentration in blood andΔDA value in extracellular fluids of striatum, between L-DOPA concentration andΔDA value in extracellular fluids of striatum.5 Synchronous pharmacokinetics study on effects of Shouwu Fang on L-DOPA in PD rats blood and extracellular fluids of striatum. Conmpared with model low dose group, the blood L-DOPA concentration of SWF+model low dose group were increased significantly at 6 time points, the concentration in striatum was decreased at 15 min after a administration. the blood AUC(o-∞) of SWF+model low dose group was increased, blood Tmax and MRT(o-t) were delayed, and striatum Tmax and MRT(o-t) were delayed too.6 Effects of Shouwu Fang on DA and its metabolic products in extracellular fluids of striatum in PD rats. After one week injection of 6-OHDA into stratum, compared with the control group, the DA in extracellular fluids of striatum significantly was decreased, the DOPAC/DA and HVA/DA were risen of model group. Compared with the model group, model low dose group and SWF +model low dose group DA basic value increased, DOPAC/DA and HVA/DA basic value decreased. After administration of L-DOPA, ip, DA level increased rapidly and then fell slowly. Compared with the model low dose group, the SWF+model low dose group DA increased more significant, andΔDA increased less.7 Effects of Shouwu Fang on hydroxyl free radical in extracellular fluids of striatum in PD rats. The target of hydroxyl free radical is total DHBA(2,3-DHBA +2,5-DHBA). The total DHBA of model group is above that of control group. Compared with model group, model low dose group and SWF +model low dose group total DHBA basic level decreased. After administration of L-DOPA, ip, total DHBA increased gradually, then fell slowly after 275min. The SWF +model low dose group DHBA increase is lowest, and lower than model low dose group significantly at 9 time points.Conclusion1 Through the combination of blood-brain microdialysis technique and HPLC-FLD and HPLC-ED, make it possible to simultaneous detection of the L-DOPA concentration in blood, L-DOPA concentration and DA and its metabolic products and hydroxyl free radical in extracellular fluids of striatum, in dialysates of freely moving rats.2 There are strong correlation between L-DOPA concentration in blood, L-DOPA concentration in extracellular fluids of striatum, andΔDA value in extracellular fluids of striatum in awake PD rats. The blood concentration of L-DOPA can reflected the concentration of L-DOPA and effects in target organ partly.3 Shouwu Fang delayed L-DOPA elimination, increased its absorption in blood, and reduced fluctuation of concentration in striarum.4 Shouwu Fang and L-DOPA synergy therapy PD. SWF can retain high level of DA in extracellular fluids of striatum for a longer time, and decreasesΔDA at the same time, and reduced the DA fluctuation. This synergy effect was closely related to SWF effects on L-DOPA pharmacokinetics.5 The hydroxyl free radical basic level in extracellular fluids of striatum was decreased when administration of low dose L-DOPA than model group. SWF can promote the decrease, have an antioxidant effect. |
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