| BackgroundThis research was commissioned by Chongqing Watson pharmaceutical co., LTD. The formulation was listed on the proprietary Chinese medicine, and approved by the State Food and Drug Administration (SFDA) of China. With functions of removing blood clot and vascular blockage, it was used in the clinical treatment of different headache symptoms. However, it always lacked the control standards of the main efficacy component, the definitive scope of the modern diseases, and the effects and its mecanism and extracts dose-effect relationship for treamting migraine. This topic research, the technology reform of Duliang soft capsule in traditional Chinese medicine (TCM) varieties, received funding from special project "major drug discovery" of ministry of science and technology (item number:2010ZX09401-306-3-4).ObjectiveIn this study, firstly, the content of main ingredients of Duliang soft capsule was detected for ensuring the quality of the experimental drμg and confirming the control indicators of efficacy material. Then using nitroglycerin-induced migraine model rats, dose-effect relationship of Duliang extracts was studied to obtain the best dosage. Finally its effect and mechanism were discussed. These studies provide a theoretical support for further pharmacology researches and clinical application.Methods1. Detection of main components content in Duliang soft capsule:using high performance liquid chromatograph, imperatorin, isoimperatorin and ferulic acid in Duliang soft capsule were detected. The determination condition of imperatorin and isoimperatorin:the mobile phase was acetonitrile-water(52:48) and UV detection wavelength was set at248nm, the flow rate was1.0mL-min-1and the column temperature was30"C. The determination condition of ferulic acid:the mobile phase was methanol-0.1%phosphoric acid (35:65) and UV detection wavelength was set at321nm, the flow rate was1.0mL-min-1and the column temperature was30℃2. Dose-effect relationship of Duliang extracts for treating migraine:90male SD rats,180-220g, randomly were divided into nine groups, namely normal group, model group, Sumatriptan group,6different doses (0.13g-kg-1、0.39g-kg-1、1.31g-kg-1、3.93g·kg-1、13.13g·kg-1、39.39g·kg-1) of Duliang extracts groups, each group included10rats. Each rat was given drug byintragastric for7days (dosage of10mL·kg-1), the normal and model group was given the volume of solvent. In addition to the normal group, each group10mg·kg-1subcutaneous injection of nitroglycerin in30min at day7, the normal and model group was given volume of physiological saline. Then building rat behavior observation during0-180min, and recorded results. Finally all rats were sacrificed after anesthetized with3.5%chloral hydrate after4h, serum and plasma samples were collected, and used to determine the levels of plasma calcitonin gene related peptide (CGRP) and endothelin (ET) by radioimmunoassay method and serum nitric oxide (NO) by ultraviolet spectrophotometry method. After that, the brain stem tissues were dissected, and used to assay the levels of serotonin (5-HT), norepinephrine (NE), dopamine (DA) by fluorometric method.3. Behavioral observation on migraine rats after Duliang soft capsule:60SD male rats,180-220g, randomly divided into6groups, namely normal group, model group, Sumatriptan group, low-, medium-, high-dose (0.44,1.31,3.93g-kg-1) of Duliang groups, each group included10rats. Each rat was given drug byintragastric for7days (dosage of10mL-kg’1), the normal and model group was given the volume of solvent. In addition to the normal group, each group10mg·kg-1subcutaneous injection of nitroglycerin in30min at day7, the normal and model group was given volume of physiological saline. Then building rat behavior observation during0-180min, and recorded results.4. Effect on blood biochemical indexes and brainstem monoamine transmitters of migraine rats after Duliang soft capsule:60SD male rats,180-220g, randomly divided into6groups, namely normal group, model group, Sumatriptan group, low-, medium-, high-dose (0.44,1.31,3.93g·kg-1) of Duliang groups, each group included10rats. Each rat was given drug byintragastric for7days (dosage of10mL·kg-1), the normal and model group was given the volume of solvent. In addition to the normal group, each group10mg·kg-1subcutaneous injection of nitroglycerin in30min at day7, the normal and model group was given volume of physiological saline. All rats were sacrificed after anesthetized with3.5%chloral hydrate after4h, serum and plasma samples were collected, and used to determine the levels of CGRP and ET by radioimmunoassay method and serum NO by ultraviolet spectrophotometry method. After that, the brain stem tissues were dissected, and used to assay the levels of5-HT, NE, DA by fluorometric method.5. Effect on inflammation relevant factors migraine rats after Duliang soft capsule:60SD male rats,180-220g, randomly divided into6groups, namely normal group, model group, Sumatriptan group, low-, medium-, high-dose (0.44,1.31,3.93g·kg-1) of Duliang groups, each group included10rats. Each rat was given drug byintragastric for7days (dosage of10mL·kg-1), the normal and model group was given the volume of solvent. In addition to the normal group, each group10mg·kg-1subcutaneous injection of nitroglycerin in30min at day7, the normal and model group was given volume of physiological saline. All rats were sacrificed after anesthetized with3.5%chloral hydrate after4h, brain stem were separated on the ice, washed in the precooling buffer, and cut tissue into small pieces. Total and nucleus protein were extracted according to kit instructions, and BCA method were used to detect the content. Finally expression level of nucleus or total protein including nuclear factor kappa B p65(NF-κB p65), cellular oncogene fos (c-fos), nitric oxide synthase (iNOS), Interleukin1β (IL-1β) and cyclooxygenase-2(COX-2) were determinate by Western Blot.Results1. Detection of main components content in Duliang soft capsule:imperatorin, isoimperatorin and ferulic acid content were1.54mg·g-1,0.49mg-g"1and51.46μg·g-1respectively.2. Dose-effect relationship of Duliang extracts for treating migraine:In terms of behavior observation, compared with normal group, model group rats in the whole observation period (0-180min) always scratched head, scratching number and climbing cage number increased significantly (P<0.01), and red ear symptoms appeared. Compared with model group, scratching number and climbing cage number of Duliang extracts0.13g·kg-1,0.39g·kg-1,1.31g·kg-1,3.93g·kg-1,13.13g·kg-1,39.39g·kg-1were not decline in0-60min, there is no significant difference (P>0.05).1.31g·kg-1,3.93g·kg-1,13.13g·kg-1,39.39g·kg-1in90-180min were decrease significantly (P<0.05or P<0.01), and disappear time of ear red and climbing cage significantly shortened (P<0.01). In terms of impact on blood biochemical indexes and brainstem monoamine transmitters, the concentration of serum NO and plasma CGRP in the dose of1.31g-kg"1,3.93g·kg-1,13.13g·kg-1,39.39g·kg-1were significantly decreased (P<0.05), and5-HT and NE in brain stem were remarkably increased. The levels of plasma ET in the dose of3.93g·kg-1,13.13g·kg-1,39.39g·kg-1and brain stem DA in the dose of39.39g·kg-1were significantly increased (P<0.01or P<0.05). All indicator presented a certain dose-effect relationship at dose of1.31g·kg-1to39.39g·kg-1.3. Behavioral observation on migraine rats after Duliang soft capsule:Compared with normal group, model group rats in the whole observation period (0-180min) always scratched head, scratching number and climbing cage number increased significantly (P<0.01), and red ear symptoms appeared, the results suggested the model was successful. Compared with model group, scratching number and climbing cage number of Duliang soft capsule,0.44g·kg-1,1.31g·kg-1,3.93g·kg-’were not decline in0-60min, there is no significant difference (P>0.05).1.31g·kg-1,3.93g·kg-1in60-180min were decrease significantly (P<0.05or P<0.01), and disappear time of ear red and climbing cage significantly shortened (P<0.01). The results suggested Duliang soft capsule could significantly improve the abnormal behavior of migraine rats during60min to180min after building model.4. Effect on blood biochemical indexes and brainstem monoamine transmitters of migraine rats after Duliang soft capsule:compared with normal group, plasma CGRP and serum NO of model group increased significantly (P<0.01), plasma ET and5-HT NE and DA levels in the brain stem were decrease significantly (P<0.01).5-HT, NE, DA content in Duliang soft capsule0.44g·kg-1group were significantly increased (P<0.01), plasma CGRP and serum NO content in1.31g-kg-1group were decreased significantly (P<0.01),5-HT, NE, DA content significantly increased (P<0.01), plasma CGRP and serum NO contents of3.93g·kg-1group were decreased significantly (P<0.01), plasma ET,5-HT, NE, DA content were increased significantly (P<0.01). The results suggested Duliang soft capsule could significantly improve the abnormal blood biochemical indexes and brainstem monoamine transmitters of migraine rats.5. Effect on inflammation relevant factors of migraine rats after Duliang soft capsule:compared with normal group, the expression level of NF-κB p65, c-fos in nucleus and iNOS, IL-1β, COX-2in total protein in model group rats was significantly increased (P<0.01). Compared with model group, the expression level of NF-κB p65, c-fos, iNOS, COX-2and IL-1β in Duliang soft capsule1.31g·kg-1,3.93g·kg-1group was significantly decreased (P<0.05or P<0.01). The results suggested Duliang soft capsule could significantly improve the abnormal inflammation relevant factors of migraine rats.Conclusion1. Success to establish testing method of main components of Duliang soft capsule and its quality control standard, and ensure the reliability and repeatability of this study.2. Success to copy nitroglycerin-induced migraine model, and applied to Duliang soft capsule in the related research.3. In the dose-effect relationship of Duliang extracts for treating migraine, Duliang extracts0.13g·kg-1,0.39g·kg-1group could improve the related indexes, but there was not significant difference. The dose of1.31g·kg-1-39.39g·kg"1improved significantly related indicators, and presented a certain dose-effect relationship.4. Duliang soft capsule in the dose of1.31g·kg-1to3.93g·kg-1could improve significantly the abnormal changes of behavior, blood biochemical indexes, brainstem monoamine transmitters and inflammation relevant factors of migraine rats.5. Duliang soft capsule could significantly improve the relevant indicators of the abnormal changes of vasomotor function and nerve pain sensitization in blood and brain tissue of migraine rats. It could be one of the important mechanisms for the treatment of migraine.6. In the brainstem of nitroglycerin-induced migraine rats, NF-κB signal was activated, which caused the expression of many inflammatory factors. It proved that nitroglycerin-induced migraine might be due to aseptic inflammaion of the brainstem caused by nitroglycerin. What’s more, Duliang soft capsule could obviously reduce the expression of these inflammatory factors, so it could also be one of the important mechanisms for treatment of migraine. |
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