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Objective:To study the metabolites of 17β-estradiol via human cytochrome enzyme CYP 1B1 and analyze the genera-tion rate of products by a high performance liquid chromatography coupled with electrochemical detector (HPLC-ECD) method.Meth-ods:A Mightysil RP-18GP (250 mm ×3.0 mm, 5 μm) column was used at the temperature of 40 ℃.The electrochemical detector with E=+900 mV was applied, the mobile phase was 0.5％NaH2PO4(pH 3.0) and methanol (45:55), the flow rate was 0.5 ml · min-1 , and the injection volume was 5μl.Results:The main metabolite was 4-OH-E2 accompanied with a little of 2-OH-E2.The average hydroxylation rate of 4-OH-E2 was about five times as much as that of 2-OH-E2 at the same concentration of estrogen E 2 me-tabolized via CYP1B1.Conclusion:Taken together, CYP1B1 catalyzed hydroxylation sites of 17-beta estradiol based on NADPH me-tabolism are maily 4.
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Objective To study the content of monoamine neurotransmitters and neurotrophic factor in the hippocampus, amygdala and prefrontal cortex in anxious depression rats, and explore the possible pathogenesis.Methods 60 SD rats were randomly divided into normal group, vehicle group, anxiety group, depression group, and anxious depression group, 12 rats in each group.Chronic restraint stress combined with corticosterone injection was used to establish anxiety and depression model, the modeling time was 21 d.After modeling, elevated plus maze test, open field test, and forced swimming test were used to evaluate the anxiety and depression-like behavior, HPLC-ECD was used to detect the content of 5-HT, NE, and DA in the hippocampus, amygdala, and prefrontal cortex of rats.Western-blotting was used to detect the expression of BDNF and NT-3 in rats.Results Rats in anxious depression model group were comparable to the anxiety group in time and frequency entering open arm time, and number of locomotor activity in open field, and it had a significant difference when compared with the control and depression groups (P<0.01 or P<0.05).Immobile time in anxious depression model rats was increased significantly when compared with the control and anxiety groups (P<0.01).Meanwhile, compared with the control group, 5-HT in hippocampus and 5-HT, NE in amygdala or prefrontal cortex were significantly decreased in the depressive rats with anxiety (P<0.01 or P<0.05).Moreover, the content of BDNF and NT-3 was significantly decreased in each brain regions compared with the control group (P<0.01 or P<0.05), and BDNF levels were obviously decreased compared with the anxiety group (P<0.05).Conclusions Rats of anxious depression have significant anxiety and depression-like behaviors.Its mechanism may be associated with the down-regulation of monoamine neurotransmitters and neurotrophic factors BDNF and NT-3 in hippocampus, amygdala, and prefrontal cortex region.
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OBJECTIVE: To determine the antioxidant activities of different extracts from Phellinus robustus (PR) and analyze the active components of the extract with the highest antioxidant activity. METHODS: PR was first extracted by ethanol and then the ethanol extract was sequentially partitioned into four fractions. To determine the antioxidant abilities of different extracts, the contents of total phenols and total flavonoids, scavenging activity on DPPH and ABTS radicals, reducing power and inhibition of lipid peroxidation were measured. RESULTS: The ethyl acetate fraction (EAF) showed the highest contents of total phenols and flavonoids and the largest antioxidant capacity, meanwhile, its DPPH radical scavenging activity was comparable to that of the positive control, ascorbic acid (P>0.05). TPC and TFC showed strong positive correlations with FRAP (r2=0.984, P<0.01). Three phenolic acids (protocatechuic acid, vanillic acid and caffeic acid) had been isolated and identified from EAF by HPLC-ECD, and anilic acid and caffeic acid were first isolated from PR. CONCLUSION: EAF from PR has strong potential to be a source of phenolic compounds with antioxidant proprieties and a promising source of food and drugs.
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Aim Toinvestigatetheeffectofhigh,me-dium and low doses of Mahuang decoction on the re-lease amount of rat hippocampal neural transmitter (Glu,Gly,Asp,GABA),then compare Mahuang de-contion with ephedra alkaloids and ephedra.Methods Ratswererandomlydividedinto6groupsandgiven orally with Mahuang decoction of high dose (calculated by ephedra 4 g·kg-1 ),medium dose (calculated by ephedra 2 g·kg-1 ),low dose (calculated by ephedra 1 g · kg-1 ),ephedra (calculated by ephedra 2 g · kg-1 ),ephedra alkaloids (ephedrine 7 mg · kg-1 , pseudoephedrine 2. 4 mg · kg-1 , methylephedrine 1. 12 mg · kg-1 )and blank control group.Samples were obtained from the hippocampus of conscious rat by microdialysis sampling technique.The content of ami-no acid neurotransmitters in dialysates was detected u-sing the established HPLC-ECD with OPA pre-column derivationmethod.Results Fouraminoacidneuro-transmitters could be well separated in 28 min.High, medium and low doses of Mahuang decoction,ephedra and ephedra alkaloids significantly increased the con-tent of these four amino acid neurotransmitters,com-pared with blank control group (P<0. 05 ).Ephedra alkaloids significantly reduced the levels of inhibitoryamino acid neurotransmitters GABA and Gly in 90 min,compared with the medium dose of Mahuang de-coction.Excitatory neurotransmitters of ASP and Glu in hippocampus showed the trend of increase first and then decrease after oral administration of Mahuang de-coction and ephedra. The levels of Glu and Asp reached peaks from 90 min to 120 min after treatment with Mahuang decoction,and also increased along with dose increase of Mahuang decoction.In comparison with the medium dose of Mahuang decoction group,the level of Glu reached peak at 90 min and 150 min in ephedra alkaloids group and ephedra group respective-ly,and the content of Glu significantly increased at peaktime.Conclusions Increasedcontentofexcita-tory amino acid neurotransmitters (Asp and Glu ) shows positive correlation with the dose of Mahuang de-coction.Other components in Mahuang decoction in-hibits the up-regulation effect of ephedra and ephedra alkaloids on Glu,and promotes the up-regulation effect of ephedra alkaloids on GABA and Gly.
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OBJECTIVE To investigate the anti- tremor effect and mechanism of baicalein on oxotremorine- induced muscle tremor in mice. METHODS The acute model of muscular tremor was induced by intraperitoneal injection of oxotremorine, and the latency, duration and frequency of muscle tremor in mice were measured immediately; the saliva of mice was measured to reflect the correlation between tremor and peripheral nerve function; the aim of this study was to determine the content of MDA and the activity of GSH-PX, and to investigate the anti-oxidation of mice with tremor model. The activity of acetylcholinesterase (AchE) and acetylcholine transferase (ChAT) can indirectly reflect the level of acetylcholine in the brain. The level of monoamine neurotransmitters in brain tissue was determined by high performance liquid chromatography (HPLC-ECD). RESULTS The animals in the model group appeared obvious tremoring, salivating and erecting and other symptoms. Compared to the model group, there was no obvious inhibitory effect on the administration of each dose. After 7, 14, 21 and 28 d of continuous administration, the latency, duration and tremor frequency of tremor mice were significantly shortened, the levels of acetylcholine were significantly decreased, the changes of DOPAC and DA neurotransmitters in the brain of model group were recovered, regulate the dynamic balance of acetylcholine and dopamine in the brain. CONCLUSION Long- term administration can improve the tremor behavior of mice, the mechanismmay be related to the regulation of neurotransmittersin brain.
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Aim To investigate the effects of diterpene ginkgolides meglumine injection (DGMI ) on amino acids and monoamine neurotransmitters in rats with cerebral ischemia/reperfusion injury.Methods In-traluminal suture was applied to establish middle cere-bral artery occlusion (MCAO/R)model with ischemia for 1.5 h and reperfusion for 24 h.After the adminis-tration of DGMI (i.v.),the levels of amino acid and monoamine neurotransmitters in brain tissue were de-tected through HPLC-ECD.Results DGMI down-reg-ulated the concentrations of aspartic acid, glutamic acid,glycine and γ-aminobutyric acid which were in-creased in MCAO/R group.DGMI also reduced the levels of norepinephrine epinephrine,glyoxylic acid, serotonin and 5-HIAA in cortex and hippocampus,and increased adrenaline content compared to the model group.Conclusion DGMI exhibits a protective role in rats with cerebral ischemia /reperfusion injury through regulating amino acids and monoamine neuro-transmitters.
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Aim To investigate the influence of echi-nacoside ( ECH ) on monoaminergic neurotransmitter extracellular of the hippocampus and cerebral cortex in rat model of Alzheimer′s disease ( AD) , and ultimately to provide a theoretical basis for ECH′s improving the ability of learning and memory. Methods 60 male SD rats were randomly divided into 6 groups with 10 rats in each group : sham operation group, model, ECH groups of low, medium and high doses (10, 20, 40μg ·g-1 ·d-1 ) , and Hup A ( Huperzine A, 0. 02 μg· g-1·d-1) group. The AD rat model was established by abdominal cavity injection with D-galactose and uni-laterally injected with amyloid beta-protein fragment 25-35 ( Aβ25-35 ) into the right hippocampus. Morris wa-ter maze test was used to study the animals′ ability of spatial learning and memory. The synchronous dual-probe dual-channel brain microdialysis sampling tech-nology was applied to collect dialysates from different encephalic areas continuously, and combined with HPLC electrochemical detection were used to measure the extracellular levels of norepinephrine ( NE) , dopa-mine (DA), 5-serotonin (5-HT). Results 1. Com-pared with the sham operation group, the mean escape latency of the model group was significantly prolonged ( P<0. 05 ) , and the time that rats were in the plat-form quadrant was significantly shortened ( P<0. 05 );on the contrary, compared with the model group, the mean escape latency of ECH groups were significantly shortened ( P<0. 05 ) , and the time that rats were in the platform quadrant was significantly extended ( P<0. 05). 2. Compared with the sham operation group, the contents of NE,DA and 5-HT were significantly de-creased in the model group ( P <0. 05 ) . However, compared with the model group , ECH could improve the concentrations of NE, DA, 5-HT in the hippocam-pus and cerebral cortex, and these monoamine levels of the brain regions were restored to near control. Con-clusion ECH can effectively improve the ability of learning and memory of rats with AD, giving a rise to the monoamine neurotransmitter both in hippocampus and cortex, exerting a positive effect on treatment of cognitive dysfunction . The ECH low dose group is sig-nificantly lower than the ECH groups of medium and high doses and Hup A group in improving the ability of learning and memory.
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OBJECTIVE: To examine the effects of echinacoside (ECH) on striatal extracellular levels of hydroxyl radical in cerebral ischemia rats and its possible mechanism of anti-cerebral ischemia. METHODS: Rats were divided into control, model, ECH high and low dose and CXQ groups randomly. Every rat was administered drugs or vehicle through intraperitoneal injection, one time a day for 7 consecutive days. At day 3, focal ischemia was generated by permanent middle cerebral artery occlusion (MCAO). Then the striatal extracellular fluids were gained by brain microdialysis. High performance liquid chromatography with electrochemical detection was used to measure the striatal extracellular levels of hydroxyl radicals. RESULTS: The levels of 2, 3-DHBA, 2, 5-DHBA increased rapidly. Compared with the model group, administration of ECH of high and low dose (30, 15 mg · kg-1 · d-1) and CXQ (30 mg · kg-1 · d-1) successfully prevented the elevation of 2, 3-DHBA and 2, 5-DHBA. CONCLUSION: ECH can reduce striatal extracellular levels of hydroxyl radical, which may be one of its mechanisms of anti-cerebral ischemia. Copyright 2012 by the Chinese Pharmaceutical Association.
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The qualitative and quantitative analysis of active constituents in Fructus Psoraleae is presented by high-performance liquid chromatography (HPLC) coupled with different detections.Extracts of Fructus Psoraleae were examined by HPLC with ion trap mass spectrometry (IT-MS) and 18 major compounds of coumarins,benzofuran glycosides,flavonoids,and meroterpene were identified.The determination of four major constituents including bavachin,isobavachalcone,bavachinin,and bakuchiol was accomplished by HPLC with UV,MS,and electrochemical detection (ECD).These methods were evaluated for a number of validation characteristics (repeatability,LOD,calibration range,and recovery).ECD obtained a high sensitivity for analysis of the four components; MS provided a high selectivity and sensitivity for determination of bavachin,isobavachalcone,and bavachinin in negative-ion mode.After optimization of the methods,separation,identification.and quantification of the four components in Fructus Psoraleae were comprehensively tested by HPLC with UV,MS,and ECD.
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OBJECTIVE:To develop a HPLC-ECD method for the determination of scutellarin in human plasma.METH-ODS:The plasma sample injection was performed after protein precipitation by methanol.The mobile phase consisted of 0.083mol? L-1 sodium dihydrogen phosphate-methanol-acetonitrile-tetrahydrofuran(60:30:10:0.4),which was added dropwise with H3PO4 in a ratio of 100:106).The column temperature was under room temperature;the detection voltage was 100mV;the protection voltage was 500mV;the flow rate was 1mL? min-1 and the sample size was 20? L.RESULTS:The calib-ration curve was linear in the range of 20.0～ 400.0?g? L-1(r=0.999 9).The methodological recovery was 96.79% ～ 108.50%.Both the intra-day and inter-day RSD were less than 5%.CONCLUSION:This HPLC-ECD method is accurate,sensitive and simple,and suitable for pharmacokinetic study of scutellarin.
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AIM: To develop a sensitive and rapid determination method of dopamine(DA) and its metabolites 3,4-dihydroxyphenylacetic acid(DOPAC) and homovanillic acid(HVA) in rat cerebral microdialysates.METHODS: Microdialysis probes were placed into the right stratums of Wistar rat brains and perfused with Ringer's solution at a rate of(1.5)(?l?min~(-1)).A reverse phase HPLC with electrochemical detection (ECD) were used to assay DA,DOPAC and HVA after the cerebral microdialysates were collected every 20 minutes from awake,freely moving rats.In order to know the reliability of this method,selectivity,linear range,precision and accuracy were tested and the contents of DA,DOPAC and HVA in rat microdialysates were determined.RESULTS:The standard curve were in good linear at the range of(12.5) to 250(?g?L~(-1)).The recovery rates of DOPAC,DA and HVA at the concentration of(0.05),(0.13),(0.25)(?g?L~(-1)) were(98.33)%,(102.67)%,(92.33)% respectively.Their with-in day RSD were(3.3)%,(3.4)%,(2.5)% and between-day RSD(4.2)%,(2.3)%,(5.6)% respectively.The contents of DOPAC,DA and HVA in rat microdialysates were(1.79)?(0.07),(0.45)?(0.02),(1.67)?(0.05)(?g?ml~(-1)) (n=6),respectively.CONCLUSION: The simple,accurate and stable method can be applied to the study of the basic researches of diseases related to DA by cerebral microdialysis in rat.
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AIM To determine the dynamic changes of monoamine neurotransmitters and their metabolites in various brain regions of cerebral ischemia reperfusion mice. METHODS Concentrations of monoamine neurotransmitters such as norepinephrine (NE), dopamine (DA), serotonin (5-HT) and metabolites were determined by HPLC-ECD on d 0,1,3,5 and d 20 after cerebral ischemia reperfusion by common carotid artery occlusion. RESULTS The cerebral ischemia reperfusion mice showed decreased concentrations of NE, MHPG, DA, DOPAC, 5-HT and 5-HIAA in various brain regions, especially in hippocampus. CONCLUSION Several neuron systems play an important role in neurons damage of cerebral ischemia reperfusion, especially the NE and DA in hippocampus which is sensitive to the ischemia damage. The data offer useful guides for clinical treatments of cerebral ischemia diseases.
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Objective To study the effect of GnRH on the secretion of 5-HT in the stomach. Methods The GnRH-A Alarelin was directly injected into the stomach of rats to observe the change of density of 5-HT immunoreactive positive cell in the stomach and small intestine by immunohistochemical SABC method and 5-HT level in the circulating blood by HPLC-ECD, Results Compared with the control group,which was injected with saline into the stomach,the density of immunoreactive positive cell was significantly increased in the stomach and small intestine in the GnRH-A treatment group;while 5-HT level in the circulating blood was significantly reduced in the experimental group.Conclusion GnRH can inhibit the releasing of 5-HT but not change its synthesis.
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Changes in the levels of biogenic amines in different brain regions and the cerebrospinal fluid in rats were measured after acute or chronic treatment with tricyclic antidepressants. After single or 3 weeks' treatment with imipramine or desipramine, blocks of tissues were obtained from seven regions of the brain (frontal cortex, corpus striatum, hippocampus, thalamus, hypothalamus, substantia nigra and cerebellum) immediately after collection of the cerebrospinal fluid (CSF) from the cisterna magna. The concentrations of biogenic amines and their metabolites (norepinephrine, epinephrine, dopamine, 5-hydroxytryptamine (5-HT), dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), 5-hydroxyindoleacetic acid (5-HIAA)) in brain tissues and the CSF were measured using the high performance liquid chromatography-electrochemical detection system (HPLC-ECD). Treatment with desipramine or imipramine caused major alterations in the concentrations of central norepinephrine or 5-HT and its metabolite, respectively. Brain regional responses were variable according to the kind of tricyclic antidepressants and the duration of treatment. It is noteworthy that chronic treatment with both desipramine and imipramine caused altered hippocampal concentrations of norepinephrine and/or 5-HT and its metabolites. Striatal DOPAC concentrations were also changed after acute or chronic treatment with both drugs. These results suggest that tricyclic antidepressants altered neurotransmission according to the brain region, and the hippocampal norepinephrine and 5-HT and/or the striatal dopamine may have a significant role for the expression of antidepressant action of tricyclic antidepressants.
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Masculino , Ratos , Animais , Antidepressivos Tricíclicos/farmacologia , Monoaminas Biogênicas/metabolismo , Encéfalo/efeitos dos fármacos , Desipramina/farmacologia , Imipramina/farmacologia , Ratos Sprague-DawleyRESUMO
The selective vulnerability of the basal ganglia to carbon monoxide(CO) is well recognized. And hyperbaric oxygen therapy(HBO) has long been accepted as a primary treatment for CO poisoning; however, the mechanism of action and objective methods to assess the therapeutic efhcacy of HBO have not been fully established. To evaluate the effect of HBO on the neurotransmitter changes in CO poisoning the striatal dopamine and its metabolites were measured in CO-intoxicated and HBO treated rats by high performance liquid chromatograpy with electrochemical detection. Male SpragueDawley rats, weighing 250-350 g were exposed to 4,500 ppm CO for 30 minutes and a group of them was treated with HBO(3 ATA, 30 miutes). The neostriatum of the rats were obtained and investigated according to the various time lapse after the cessation of CO exposure or the completion of HBO, The results are as follows: 1. The application of the routine HBO to the normal rat did not affect the levels of striatal neurotransmitters. 2. The concentration of striatal DA was markedly increased in CO-intoxicated rat during acute phase and it was reversed by HBO. But the effect did not last more than 24 hours. 3. In the delayed phase of experiment, the concentration of striatal DA tended to be decreased, which was partially thought to be due to the striatal injury, damage of striatonigral pathway or neuronal loss of substantia nigra by C0-intoxication.These results suggest that dopaminergic system might be implicated both in the pathogenesis of C0-induced striatal injury and the therapeutic efficacy of HB0.