| Cerebral vascular diseases are harmful to human life and heath.Ischemia cerebrovascular disease (ICVD) is a common disease inmiddle-aged and elderly people. Its characteristic includes high diseaseincidence rate,high prevalence rate,high recurrence rate,high cripplingrate and high mortality rate. ICVD is one of key diseases that have beenprevented or treated at home and abroad. So a lot of human and materialresources have been put into the basic and clinical study. Up to now theprimary pathological mechanisms and causes of ICVD have not beendefinite. As the lack of effective diagnostic and therapeutic methods,it isvery important and urgent to study the drug for ICVD. In recent years,inorder to find better ways and better drugs,domestic and foreignresearchers have done a lot of researches in treating ICVD with the activeingredients of natural medicines,but there is no satisfactory therapy forICVD.Chrysophanol is the compound from rhubarb anthraquinones. Theprevious study confirms that it has an evident anti-aging effect. As thesolubility of chrysophanol in water is low,its nature is unstable and thestimulating effect on gastrointestinal is obvious when used directly,itsfurther clinical application is limited. In previous studies,chrysophanol'ssolvent is mainly 10 % N, N-dimethylformamide (DMF). Because it isorganic solvent and has harmful effect,it can't be used in clinicaladministration. But it can be used in the experiment at low concentrationand small dosage. In order to find new and suitable dosage forms forclinical application,we conducted screening study on clinical pharmacology research of chrysophanol formulations. Currently,chrysophanol was made into nanocapsules loading with chrysophanol bypolybutylcyanoacrylate,chrysophanol hydroxypropyl-β-cyclodextrininclusion complex and chrysophanol liposomes by the best preparationmethod and technics,and their preliminary pharmacokinetic studies werecompleted. In order to screen out the best formulation for clinicalapplications,we compared the differences of organization distribution andpharmacodynamics effects in three chrysophanol formulations by adoptingto cerebral ischemia reperfusion.285 male mice were divided into nineteen groups randomly:Group 1were normal control group,sham-operated group,cerebral ischemiareperfusion model control group,and the solvent control group of DMF,nanocapsules,inclusion complex and liposomes；Group 2 werechrysophanol (10 % DMF dissolved) 10.0,1.0,0.1 mg·kg-1 dose group；Group 3 were nanocapsules loading with chrysophanol by polybutylcyanoacrylate10.0,1.0,0.1 mg·kg-1 dose group；Group 4 were chrysophanolhydroxypropyl-β-cyclodextrin inclusion complex 10.0,1.0,0.1 mg·kg-1dose group；Group 5 were chrysophanol liposomes 10.0,1.0,0.1 mg·kg-1dose group.In various groups,cerebral ischemia reperfusion were administeredexcept normal control and sham-operated group. Sham group wasoperated on but not executed cerebral ischemia reperfusion. Aftermodeling,corresponding drugs were administered for every groups andnormal saline (NS) for normal group,sham-operated group,cerebralischemia reperfusion model control group,once a day for eighteen days inthe same way. The ethological experiments,about learning and memory,such as passive avoidance test (d 8~d 9),exploratory movement test (d11~d 12),step-through and step-down test (d 14~d 15) and shuttle test (d17~d 18) etc,were observed the improving effects of chrysophanolformulations on memory impairment cerebral ischemia reperfusion-induced. Through determination of the activities of CAT,GSH-Px,SOD,NO,NOS,MAO-B in brain,liver and blood tissues,the contents of MDA in brain and liver tissues,the contents of LF in brainand heart tissues,we observed the effects on oxidative related enzymes'activity and metabolites'content of chrysophanol formulations. Throughdetermination of activity of AChE in brain tissue,we observed thepharmacodynamic effects of chrysophanol formulations. Throughdetermination of contents of amino acid(AA) in brain tissue,we observedthe pharmacodynamic effects of chrysophanol formulations. Throughdetermination of survival time in beheaded hypoxia mice and brain index,we observed the pharmacodynamic effects of chrysophanol formulations.Through the TTC and HE dying experiment in brain and liver tissues,weobserved the brain's infarction area and pathomorphological changes ofchrysophanol formulations. Through determination of chrysophanolcontent in tissues by High Performance Liquid Chromatography(HPLC),we observed tissue distribution's differences of chrysophanolformulations.1 Pharmacodynamicharmacodynamics experiments of three chrysophanol formulations1.1 Effect of three chrysophanol formulations on learning andmemoryResults showed that cerebral ischemia could cause significantmemory impairment in mice (p<0.05~p<0.01). Compared with thecorresponding model group,chrysophanol DMF and chrysophanolformulations (10.0,1.0 mg·kg-1 dose groups) could improve theimpairments of memory with cerebral ischemia reperfusion:instep-through test,the latency had an extended tendency,and the number ofentry times was significantly reduced (p<0.05~p<0.01)；In exploratorymovement test,the time of climbing darkroom was extended,height andbright room was evidently brought forward and the number of climbingheight was evidently increased (p<0.05~p<0.01)；In platform test,the latency and the number of error times were significantly decreased(p<0.05~p<0.01)；In shuttle test,the number and time of electric shockwas significantly decreased,the time of initiative avoiding was evidentlyprolonged (p<0.05). Chrysophanol DMF and chrysophanol formulations(0.1 mg·kg-1 dose group) had no significant improvements in the aboveindicators (p>0.05).Compared with chrysophanol group,the two groups of nanocapsulesand inclusion complex had no significant difference in improving memoryimpairment；However,chrysophanol liposome was better in this respect,particularly in 10.0 mg·kg-1 dose group (p<0.01).These results demonstrated that chrysophanol DMF andchrysophanol formulations had different levels in improving theimpairments of memory with cerebral ischemia reperfusion and the bestformulation was chrysophanol liposomes.1.2 Effect of three chrysophanol formulations on oxidative relatedenzymes'activity and metabolites'content in tissuesResults showed that cerebral ischemia-reperfusion injury could lowerthe levels of CAT,GSH-Px,SOD in brain,liver and blood tissues(p<0.05~p<0.01),improve the levels of NO,NOS and MAO-B in brain,liver and blood tissues (p<0.05~p<0.01),increase the contents of MDA inbrain and liver tissues (p<0.01) and the contents of LF in brain and hearttissues (p<0.01). Compared with the corresponding model group,chrysophanol DMF and chrysophanol formulations (10.0,1.0 mg·kg-1dose groups) could improve the levels of oxidative related enzymes'activity and metabolites'content (p<0.05~p<0.01),which could improvethe cerebral ischemia-reperfusion injury,and these results were related tothe dose. Chrysophanol DMF and chrysophanol formulations (0.1 mg·kg-1dose group) had no significant improvements to the above indicators.Compared with chrysophanol DMF group,the nanocapsules and inclusioncomplex groups had no significant difference (p>0.05)；However, chrysophanol liposome was better in this respect,particularly in 10.0mg·kg-1 dose group (p<0.01).These results demonstrated that chrysophanol DMF andchrysophanol formulations had different levels in improving the levels ofoxidative related enzymes'activity and metabolites'content and the bestformulation was chrysophanol liposomes.1.3 Effect of three chrysophanol formulations on AChE in brain tissueResults showed that,cerebral ischemia-reperfusion injury couldimprove the activity of AChE in brain tissue (p<0.01). Compared with thecorresponding model group,chrysophanol DMF and chrysophanolformulations (10.0,1.0 mg·kg-1 dose groups) could lower the activity ofAChE (p<0.05~p<0.01),and the results were related to the dose；Compared with chrysophanol DMF group,the two groups of nanocapsulesand inclusion complex had no significant difference in the activity ofAChE (p>0.05)；However,chrysophanol liposome was better in thisrespect,particularly in 10.0 mg·kg-1 dose group (p<0.01).These results demonstrated that chrysophanol DMF andchrysophanol formulations had different levels in improving the activity ofAChE and the best formulation was chrysophanol liposomes.1.4 Effect of three chrysophanol formulations on amino acid in braintissueResults showed that,cerebral ischemia-reperfusion injury couldlower the contents of AA in brain tissue (p<0.05). Compared with thecorresponding model group,chrysophanol DMF and chrysophanolformulations (10.0,1.0 mg·kg-1 dose groups) could improve the contentsof AA in brain tissue (p<0.05~p<0.01),and the results were related to thedose；Compared with chrysophanol DMF group,the two groups ofnanocapsules and inclusion complex had no significant difference in thecontents of AA (p>0.05)；However,chrysophanol liposome was better inthis respect,particularly in 10.0 mg·kg-1 dose group (p<0.01). These results demonstrated that chrysophanol DMF andchrysophanol formulations had different levels in influencing the contentsof AA in brain tissue and the best formulation was chrysophanolliposomes.1.5 Effect of three chrysophanol formulations on hypoxia toleranceResults showed that,cerebral ischemia-reperfusion injury couldshorten the survival time of beheaded hypoxia,the number of mouth andbrain index (P<0.05). Compared with the corresponding model group,chrysophanol DMF and chrysophanol formulations (10.0,1.0 mg·kg-1dose groups) could prolong the survival time of beheaded hypoxia,thenumber of mouth and brain indexed (P<0.05),and the result were relatedto the dose；Compared with chrysophanol DMF group,the two groups ofnanocapsules and inclusion complex had no significant difference(p>0.05)；However,chrysophanol liposome was better in this respect,particularly in 10.0 mg·kg-1 dose group (p<0.05).These results demonstrated that chrysophanol DMF andchrysophanol formulations had different levels in influencing the effects ofhypoxia tolerance and the best formulation was chrysophanol liposomes.1.6 Effect of three chrysophanol formulations on pathomorphology inbrain and liver tissuesResults were as follows,cerebral ischemia reperfusion injury haddifferent levels of pathological changes in brain and liver tissues. Incontrast to the corresponding model group,chrysophanol DMF andchrysophanol formulations (10.0,1.0 mg·kg-1 dose groups) could decreasecerebral infarction size (P<0.01),neurons'density and neuronal structureof nerve cells in hippocampal area were normal,the interspace among thenerve cells were decreased,the number of nerve cells increased and thecircumscription between cell nucleus and membrane of some nerve cellswere clear,lobule boundary,hepatic cell structure and arrangement ofwere also normal,the number of hepatic cells increased,and the results were related to the dose；In contrast to chrysophanol DMF group,the twogroups of nanocapsules and inclusion complex had no significantdifference (p>0.05)；However,chrysophanol liposome was better in theserespects,particularly in 10.0 mg·kg-1 dose group(p<0.01).These results demonstrated that chrysophanol DMF andchrysophanol formulations had different levels in influencing the effects ofthe pathological form and the best formulation was chrysophanolliposomes.2 TissuTissue distribution of three chrysophanol formulationsResults showed that,the order of chrysophanol concentration inmice's tissues by HPLC:blood>brain>kidney>liver>heart>spleen>lungs,compared with chrysophanol DMF,chrysophanol concentration ofchrysophanol formulations (10.0,1.0,0.1 mg·kg-1 dose groups) in brain,liver and blood tissues were a significant difference (p<0.05~p<0.01),andthe results were related to the dose；Compared with chrysophanol DMFgroup,the two groups of nanocapsules and inclusion complex had nosignificant difference (p>0.05)；However,chrysophanol liposome wasbetter in this respect,particularly in 10.0 mg·kg-1 dose group (p<0.01).These results demonstrated that chrysophanol DMF andchrysophanol formulations had different levels in influencing the tissuedistribution,among them,the tissue distribution of blood and brain washigher and the best formulation was chrysophanol liposomes.In summary,the cerebral ischemia reperfusion could result insenescence. The learning and memory experiments,antioxidant enzymeassays,beheaded hypoxia,cerebral infarction size and pathologicalmorphous experiments had been studied. The concentrations of chry in thedifferent tissue were determined by HPLC. All the results implied thatchrysophanol formulations could lower the contents of MDA,LF and theactivities of NO,NOS,MAO-B,improve the activities of CAT,GSH-Px,SOD,inhibit the activity of AChE,affect the level amino acid in brain, enhance the effect of hypoxia tolerance,and improve the pathologyprocess of disease. Among these chrysophanol formulations,chrysophanolliposomes was best,so it might become one of new drugs of treatingICVD in the future,in particular,the injectable dosage forms can beadministered in various routes.
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