| Migraine is a devastating neurovascular disorder characterized by mostly unilateral throbbing and pulsating head pain with a host of neurological symptoms including nausea, vomiting, photo- and phonophobia, and various autonomic, cognitive, emotional and motor disorder. Until now, the pathogenesis of migraine is still unclear. The existing theories about migraine contain vessel source theory, nerve source theory, trigeminal and vessel theory, biochemical factors, heredity factors and so on. The development of antimigraines has made great progress. The existing antimigarines contain non-steroidal anti-inflammatory drugs, ergot class, triptan and opiates. Meanwhile, some dopamine receptor antagonists, Magnesium and some natural drugs were also used to treat migraine. However, the existing antimigarines still exists various disadvantages. Exploring new antimigraines is extremely urgent.Traditional Chinese Medicines(TCMs) have been used for the treatment of pain for thousands of years. Due to their efficacy and low toxic and side effects, many reasearchers have paid attention to exploring analgestic in TCMs. Yuanhu Zhitong prescription(YZP),composed of Rhizoma Corydalis and Radix Angelicae Dahuricae, is a frequently used analgestic in China. Tetrahydropalmatine(a dopamine D2 receptor antagonist) wasconsidered as the key components of YZP. It has been used for the treatment of migraine. However, the effective ingredients and mechanism of YZP in treating migraine is still unclear.Under the guide of the theory of TCM, doctors of TCM are good at using formula of TCM to treat diseases. TCMs contains a variety of ingredients. To exploring which ingredients are the active ingredients is the main work of the TCMs researchers. All drugs play their efficacy through acting with human body. After oral administration of TCM, only the components of TCM absorbed into blood and further into the tissues may serve as active principles that have a therapeutic effect.In the present study, we choose YZP as the research object. Through pharmaceutical analysis, pharmacokinetics study, tissue distribution, behavioristics and molecular biology technique, the present study has studied the active ingredients and mechanism of YZP in treating migraine.1 Objective 1.1 Establish the chemical analysis methods YZP, and explore main ingredients of YZP. 1.2 Establish the analysis methods of YZP in plasma and brain tissues of rats, and investigate their pharmacokinetic parameters and brain tissue distribution characteristics of potential active ingredients. 1.3 Preliminary clarifying the mechanism of YZP and it’s main ingredients in treating migraine indused by nitroglycerin(GTN)2 Methods 2.1 Explortion of main ingredients of YZP 2.1.1 Sample production method of YZP After testing 50%, 75%, 95% methanol water solution in extraction, the present research found the best sample production method finally. 2.1.2 Analysis methods for YZP The present research adopted an HPLC method to analyse ingredients in YZP. Chromatographic condition: Column: Phenomenex Luna-C18 column(250×4.6 mm, 5.0 μm); The mobile phrase: AC and PT5.6; Gradien elution program: 0~15min, 23%AC; 15~26min, 23~40% AC; 26~65min, 40~65% AC; Injection sample volume: 10 μL; Detective wave length: 280 nm; Flow rate: 1 m L/min; Column temperature: 30 °C.2.2 Chemical analysis of plasma and brain tissue samples 2.2.1 Extraction method for YZP The same with 2.1.1. 2.2.2 Preparation of plasma and brain tissue samples After an oral administration of extraction of YZP, about 0.4 m L blood was collected from retinal vein plexus at 0,0.5,1,2,3,4,5,6,7,8,10,12 h. The blood was anticoagulated and then centrifuged at 3500 r/min, 4°C for 10 min. The plasma samples were abtained. After an oral administration extraction of YZP, brains were immediately collected from rats at 0, 2, 4, 6, 8, 10 h. Cortex, cerebellum, brain stem, diencephalon, striatum and hippocampus were separated rapidly on ice and collected into tubes. After weighted, they were homogenized in physiological saline solution(5-fold tissue weight) separately by a homogenizer. Then the obtained region homogenates were centrifugated separately at 12000 r/min, 4°C for 10 min and supernatant of each tissue was stored at-80°C until analysis. 2.2.3 Pretreatment methods of plasma and brain tissue samples 200 μL of plasma and brain tissue samples was mixed with 20 μL of internal standard solution in a glass tube. 2 m L ethyl acetate-light petroleum(1:1) was added into the tube, and then the mixture was vortex-mixed for 5 min. The obtained mixture was centrifuged at 2000 r/min for 10 min. The extract liquor was collected and dried in a Pressure Blowing Concentrator at 40 °C. 2.2.4 Analysis methods for plasma and brain tissue samples Chromatographic condition: Column: Phenomenex Luna-C18 column(250×4.6 mm, 5.0 μm); The mobile phrase: AC and PT5.8(51:49); Injection sample volume: 20 μL; Detective wave length: 280 nm for plasma, 230 nm for brain tissue; Flow rate: 1 m L/min; Column temperature: 30 °C. 2.3 Behavioristics study of YZP in treating migraine 2.3.1 Mechanical allodynia Mechanical allodynia was assessed by measuring incidence of foot withdrawal in response to mechanical indentation of the plantar surface of rat hind paw with von Frey fibers. 2.3.2 Thermal hyperalgesia Thermal hyperalgesia was determined by measuring foot withdrawal latency to heat stimulation using a thermal stimulator. 2.4 The effect of YZP on vasoactive substances in treating NTG indused migraine animal model The concentration of NO was determined by a one-step method. Theconcentration of 5-HT, CGRP, HIS were determined by ELISA method. 2.5 The effect of YZP on relative substances of brain in treating NTG indused migraine animal model The effect on c-fos, 5-HT, CGRP, n NOS were obtained by Western Blot or Immunofluorescence staining.3 Results 3.1 Explortion of main ingredients of YZP The research adopted a validated HPLC method for the detection of main ingredients of YZP. 12 ingredient(PTP, AYE, CTE, XTL, PME, DDE, GCE, THP, TDE, IMP, CDE, ISO)were quantitative analysis. Among these ingredients, DDE, GCE, THP, IMP were taken for the main ingredients of YZP. 3.2 Chemical analysis of plasma and brain tissue samples 3.2.1 Chemical analysis of plasma The obsorbed ingredients could be separated by the validated HPLC method. The pharmacokinetics study of the obsorded ingredients showed that THP and GCE had the highest Cmax and AUC0~∞ in the plasma(P<0.05). Among the alkaloids, the value of Cmax and AUC0~∞ of THP and GCE were higher than CDE(P<0.05). Among the coumarins, the value of Cmax and AUC0~∞ of IMP was higher than ISO(P<0.05). The value of Cmax, AUC0-∞, T1/2z, MRT0-∞ of alkaloids were higher than that of coumarins. This may suggested that Rhizoma Corydalis was the main drug in YZP. According to the results, THP, GCE and IMP may be the main ingredients of YZP. 3.2.2 Chemical analysis of brain tissue samples The obsorbed ingredients could pass through the blood-brain barrier and also could be separated by the validated HPLC method. The brain distribution study showed that the value of Cmax of THP and GCE were higher than the other ingredients(P<0.05) in all parts of the brain. THP had the highest AUC0-∞ value among these ingredients. The AUC0~∞ values of GCE and THP in the striatum were significantly greater than those in the cortex, cerebellum, brain stem, and diencephalon(P<0.05). According to the results, THP and IMP may be the main ingredients of YZP in treating migraine. 3.3 Behavioristics study of YZP in treating migraine YZP and it’s main ingrediens could improved the mechanical allodynia thermal hyperalgesia of the NTG indused migraine animal model. YZP’s effect lasted longer than the fomular of main ingrediens, while the fomular of main ingrediens took effect more quickly than YZP and had better effect than main ingrediens using separately. Working on the dopamine D2 receptormaight be the main mechanism of the above therapeutic action of YZP and the fomular of main ingrediens. 3.4 The effect of YZP on vasoactive substances in treating NTG indused migraine animal model Campared to model group, YZP and it’s main ingrediens could increase the concentrition of 5-HT, and decrease the concentration of NO,CGRP,HIS in blood. Working on the dopamine D2 receptor maight be the main mechanism of the therapeutic on 5-HT, NO,CGRP. 3.5 The effect of YZP on relative substances of brain in treating NTG indused migraine animal model Campared to model group, YZP and it’s main ingrediens increase the number of positive cells of 5-HT, and decrease the expression level of c-fos and the number of positive cells of CGRP, n NOS in caudal part Ventrolateral periaqueductal gray(VLPAG). Working on the dopamine D2 receptor maight be the main mechanism of the above therapeutic action.4 Conclusion 4.1 The validated HPLC methods were proved to be suitable for the analysis of ingredients of YZP in vivo and vitro. Combined with the results of my master degree paper, tetrahydropalmatine and imperatorin may be the main ingredients of YZP. 4.2 For the fist time, the present method proved that YZP could treat NTG indused migraine in rat through increasing and decreasing the level of migraine related molecular(increasing the level of 5-HT in the blood of jugular vein and VLPAG, decreasing the level of NO,CGRP,HIS in the blood of jugular vein, and decreasing the level of c-fos, CGRP, n NOS in VLPAG), and improved the the mechanical allodynia thermal hyperalgesia. Working on the dopamine D2 receptor maight be the main mechanism of the above therapeutic action. 4.3 The composition of YZP is very complexed. The effective ingredients and mechanism study of YZP are still needing further research. |
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