| ObjectiveThis study was to explore the differences between two kinds of uncaria alkaloids inhypnosis and neuroprotection, providing guidance for the clinical application of Uncaria.Method.①Optimizing the method for determinating monoamine neurotransmitters andrelated substances by HPLC-FLD,we established "quantitative analysis ofmulti-components by single-marker" method to determinate brain neurotransmitterssimultaneously:norepinephrine (NE), epinephrine (E), tyrosine (Tyr), dopamine (DA),3,4-dihydroxyphenyl acetic acid (DOPAC), tryptophan (Trp),3-methoxy-4-hydroxyphenylglycol (MHPG)5-hydroxytryptamine (5-HT),5-hydroxy indole acetic acid (5-HIAA),homovanillic acid (HVA), and finished the methodological study.②We gived different doses of two types of Uncaria7d(80,40,20mg/kg totalalkaloids) to KM mice,then we conducted the autonomic activities and subthreshold dose ofsodium pentobarbital (60mg/kg) sleep test.After then,we taked out the mouse whole brainor the hippocampus, striatum, cortex regions to determinate ten kinds of neurotransmitterlevels using "quantitative analysis of multi-components by single-marker" method.③We used calcium deposition method to extract liver microsomes, BCA methods todetermine the protein quantity and reduction method to determine the content of thecytochrome P450enzyme and cytochrome b5in liver microsomes (protein concentrationwas about10mg/mL). ④The PC12cells were pretreated two types of Uncaria total alkaloids for hours, thenwe added glutamate (24mmol/l) or hydrogen peroxide (180μmol/l) to co-culture, MTTmethed to determine the cell viability for studying the pre-protective effects of Uncariaalkaloids in neuron oxidative damage.Results①Under the optimized conditions,we established a method to determine ten kinds ofneurotransmitters,The calibration curves were linear in the range of0.1~2463.2ng/ml (r=0.9991), the limit of detection were between0.3~2.4ng/ml, the average recoveries werebetween93.28%~97.39%, and the relative standard deviations were between0.67%~5.01%, the methed is simple and essay to operate with good separation,low detection limitand high recovery;In a certain linear range, the RCF of NE, E, Tyr, DOPAC, Trp,MHPG,5-HT,5-HIAA, HVA to DA was1.44,1.17,1.08,0.50,0.97,5.9,3.66,3.43,1.00.The relative correction factors(RCF) had a good reproducibility (RSD=1.47%~4.89%)and no significant differences between the quantitative results by the above two methodswere observed.②0910Uncaria alkaloids could reduced locomotor activity in mice (P <0.01) andincreased the number of falling asleep in the threshold dose of pentobarbital (P <0.001),and significantly reduced the levels of DA, NE and accelerate their metabolism and orconversion rate in the hippocampus,cortex,increased the levels of5-HT, reduced theconversion rate and accelerated its generation in the hippocampus, striatum, thereby theycould promote hypnotic effect;0904Uncaria alkaloids could reduced locomotor activity inmice (P <0.01) and had no effect on the number of falling asleep in subthreshold dose ofsodium pentobarbital test in mice, and they significantly increased the level of NE, DA,reduced conversion rate and accelerated the production rate in the cortex, hippocampus,reduced the levels of5-HT in the cortical, thereby they could reduce the awakening andshowed significant effects of inducing hepatic drug metabolizing enzyme (P <0.01).③0910Uncaria alkaloids have no liver enzyme inducing effect (P>0.05),0904Uncaria alkaloids showed significant effects on inducing hepatic drug metabolizing enzyme (P <0.01).④0904Uncaria alkaloids have significant protective effect on glutamate andhydrogen peroxide-induced damage in PC12cells (P <0.001),0910alkaloids only couldhave significant protective effect on H202-induced damage in PC12cells (P <0.001).Conclusion:The established "quantitative analysis of multi-components by single-marker" methodcan be used to quantitative analysis the monoamine neurotransmitters and relatedsubstances; The reason for the difference between two types of Uncaria alkaloids in thehypnotic effect of sodium pentobarbital is at least partially attributed to hepatic drugmetabolism enzymes and brain neurotransmitters such as norepinephrine, such as dopamin5-serotonin and so on; two types of Uncaria alkaloids have a protective effect on nervecells, but their roles are differences, and it can provide the basis for the treatment ofcerebral ischemia, Alzheimer’s disease and epilepsy furtherly. In addition,0910and0904Uncaria alkaloid alkaloid may have the potential protective effects in Alzheimer’sdiseas,cerebral ischemia, depression and other diseases.These results indicate that the differences of two types of Uncaria in nervous systemshould be noted in clinical use. |
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