| Gastrodia and Uncaria are commonly used combination in TCM clinic.The combination has the effects of “calm the liver and suppress yang” and “relieving convulsion and spasm”.And it is commonly used to treat vascular migraine and Liveryang-hyperactivity migraine.The multi-component Chinese medicine is developing from combination of effective substances in various levels(effective fractioncomponents-monomer)of compound medicine.It was first proposed by academicians Boli Zhang and YongyanWang in 2005 and has become a hot spot in the research of compatibility of traditional Chinese medicine prescriptions now.Parishin A and isorhynchophylline is the active monomer component extracted from Gastrodia and Uncaria,respectively.Previous study of our research group had proved that both of them were components of Gastrodia-Uncaria drug pair that can be absorbed into the blood and can exert pharmacological activity on migraine model rats.Our research group is currently studying whether the combination of parishin A and isorhynchophylline monomer components can be used to replace the Uncaria and Gastrodia combination.Because the monomer components were more controllable,and may achieve the purpose of taking less dosage,having clearer efficacy and more controllable toxicity.So,the possibility of developing new drugs can be further studied.However,the mechanism of compatibility of monomer components is the key problem that must be solved in current research.What is the metabolic process of parishin A-isorhynchophylline and how is it distributed in vivo? What are the advantages of coadministration compared to dosing alone? What are the endogenous components related to migraine in vivo? These are the key problems for further development and clinical application.In this research,we mainly studied the metabolic process of palisarin A and isorhynchophylline in rats and its effects on migraine model rats from two aspects of ADME and lipidomics.1.The establishment of HPLC-MS/MS method for determination of parishin A and isorhynchophylline in biological samplesTo rapidly and accurately analyze the content of parishin A and isorhynchophylline in biological samples,two complete pharmacokinetic methods were established in this study.The multiple reaction detection mode of AB Sciex Triple QuadTM 4500 was applied for quantitative detection of the compounds in samples.Isorhynchophylline(m/z 384.8-241.2)and its metabolite rhynchophylline(m/z 384.8-160.2)was detected in positive ion mode,pioglitazone hydrochloride(m/z 357.1-134.1)was set as internal standard.Parishin A(m/z 995.1-726.9)and its metabolite gastrodin(m/z 331.1-123.0)were simultaneously analyzed in negative ion mode with geniposide(m/z 433.0-225)as internal standard.The sample was separated by COSMOSIL C18(2.1 x 100 mm,2.6 ?m)column with a gradient run of 0.1% formic acid aqueous solution-acetonitrile.The method can obtain a beautiful symmetrical peak shape,and the intensity was higher.The methodological validation results indicated that the method had good linear relationship(r>0.9978);The intra-and inter-day accuracy and precision were within the acceptable range(0.08<RE<7.08,1.50<RSD<8.39);Matrix effect results had proved that the method cannot be affected by the matrix in the biological sample(1.71<RSD<11.52).And a good extraction recovery rate(2.30<RSD<7.17)was acquired;The stability result also satisfy the long-term,short-term,and repeated freezethaw stability requirements(1.04< RSD<11.36).2.Comparative study on pharmacokinetics of different doses of parishin A – isorhynchophyllineTo elucidate the metabolic process of the parishin A-isorhynchophylline in rats and the metabolic properties with different doses,three different doses of high(isorhynchophylline: parishin A = 60:558.6 mg/kg),medium(isorhynchophylline: parishin A = 40:372.4 mg/kg)and low(isorhynchophylline: parishin A = 20:186.2 mg/kg)were used to administered intragastrically.DAS 3.2.8 software was used to process the blood concentration data of compounds at different time points,and a complete set of pharmacokinetic parameters was obtained.In addition,Sigma Plot 10.0 was used to draw the concentration-time curves.The results showed that there was significant positive correlation between the area under the concentration-time curve(AUC(0-t))and the peak concentration(Cmax)at different doses.For isorhynchophylline and rhynchophylline,the t1/2z and Tmax values were close,and the drug-time curve trends of the two were similar.For parishin A and gastrodin,the concentration-time curve showed that there was a significant bimodal phenomenon in parishin A,indicating that the compound had a hepatoenteral cycle in vivo.The AUC(0-t)value of gastrodin indicated that parishin A was mainly metabolized to gastrodin in vivo,and the t1/2z and Tmax of the both were also close.3.Comparative study of pharmacokinetics of parishin A-isorhynchophylline in two modes of co-administration and single-administration in migraine model ratsTo further clarify the metabolic process of parishin A and isorhynchophylline in migraine model rats and the advantages of the co-administration compared to single.Migraine model rats were performed in two different ways of single-and coadministration.The results indicated that the metabolism of each compound showed great difference in the two modes of administration.The AUC(0-t),t1/2z of isorhynchophylline and its metabolite rhynchophylline in co-administration mode was significantly greater than single-administration,indicating that co-administration can promote the compound to enter the blood,increase its half-life and prolong its efficacy.In the study of parishin A and its metabolite gastrodin,it was found that parishin A was almost undetectable by two modes of administration.But,the content of its metabolite gastrodin was high in plasma.Besides,the AUC(0-t)and t1/2z in co-administration were greater than the single administration,suggesting that parishin A exerted effect by conversion to gastrodin,and co-administration can promote its efficacy in vivo 4.Comparative study on the tissue distribution of Parishin A-isorhynchophylline in normal and migraine model ratsIn order to study the tissue distribution of parishin A-isorhynchophylline in rats,and the difference of distribution in normal rats and migraine models,Parishin Aisorhynchophylline was administered at a medium dose.After the dosing,the compouds concentrations in the heart,liver,spleen,lung,kidney,small intestine and brain of the rats at 0.5 h,1 h and 6 h were detected.The results demonstrated that isorhynchophylline and rhynchophylline were distributed in various tissues,isorhynchophylline was higher in brain,kidney and lung tissues,rhynchophylline was higher in liver,spleen and small intestine.Parishin A and gastrodin were mainly distributed in the small intestine,and almost cannot be detected in other tissues.By comparing the distribution of parishin A-isorhynchophylline in brain tissue between normal group and model group,we found that the content of isorhynchophylline and gastrodin in model group was higher than that in normal group 1 h after administration,suggesting that the pathological state of migraine may promote the content of components in the focal zone.5.Study on migraine-associated lipid effects of Parishin A-isorhynchophyllineTo find migraine-associated lipid biomarkers and study the regulation effect of Parishin A-isorhynchophylline on migraine,lipidomics techniques were used for the study.Dionex Ultimate 3000(UHPLC)Thermo Orbitrap Elite was applied for the detection of relevant lipids and Waters UPLC? RBEH C18(1.7 ?m 100*2.1 mm)column was used for separation.The data was processed by Agilent Masshunter Qualitative Analysis B.08.00 software and XCMS package under R software platform combined with SIMCA-P13.0 and SPSS statistics 21 analysis.16 important lipid biomarkers were identified,these included phosphatidylcholines(PCs),lysophosphatides(LysoPC and LysoPE),and sphingolipids(Cer and SM).In addition,PI had been proved has a significant regulatory effect on migraine-related endogenous lipid components. |
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