The Study Of Quality Control And Dose-Effect Relationship Of Tibetan Medicine Rannasangpei Pill

Qishiwei Zhenzhu pill is also named“Rannasangpei”,which is one of the most representative rare treasures of Tibetan medicine.Rannasangpei pill is made from the Pteria martensii?Dunker?,Santalum album L.,Dalbergia odorifera T.Chen,Nine stone,Crocus sativus L.,Bos taurus domesticus Gmelin,Moschus moschiferus L.and other ingredients,and is currently contained in Chinese Pharmacopoeia?2015edition,Part I?.The Rannasangpei pill boasts of effects such as tranquilization,sedation,clearing and activating the channels and collaterals,reconcile of qi and blood,and waking brain as well as a remarkable cure for the"Hei Bai Mai Bing"and"dragon blood",stroke,paralysis,hemiplegia,epilepsy,cerebral hemorrhage,cerebral concussion,heart disease,hypertension,and neurological disorders.But Rannasangpei pill formula is complex and confidential,with the chemical composition of the research focusing more on minerals and heavy metal elements while the research on plant medicine chemical composition is rarely conducted,thus hindering the improvement of the quality standards.In addition,Problems such as the dose of Rannasangpei pill spanning from a broad spectrum in clinical practice and lack of scientific evidence often result in high medicine cost and even pose a threat to patients.Objective:1.Establish the methods for quality control of plant medicine chemical composition in Rannasangpei pill;2.Reveal the dose-effect relationships of Rannasangpei pill for the cerebral ischemia-reperfusion injury on rats.Methods:1.To determination the content of composition in plant medicine,at the column temperature at 30?,the flow rate of 0.8 m L·min^-1 and the detection wavelength was at 254 nm,the mobile phase consisted of acetonitrile and 0.2%phosphate solution was used as gradient elution in Rannasangpei pill by HPLC method.2.To carry out the acute toxicity experiment of rat,and try to find out the maximum dosage,thus offering the reference in the study of dose-effect relationship.3.Set the nine doses of Rannasangpei pill,and study dose-effect relationship with focal cerebral ischemia reperfusion injury model?MCAO?in rats;Application of Thermo Kinetica Version software for data fit with nine doses and cerebral infarction rate,computation parameter of dose-effect relationship.Results:1.The six chemical components precision,repeatability,stability and the sample recovery rate all meet the requirements of Gallic acid,Corilagin,Agarotetrol,ellagic acid,Crocin I and Crocin?in Rannasangpei pill,RSD value is less than 3%,the linear range was 1.41⁴2.24?g·m L^-1?0.61¹8.24?g·mL^-1?0.30⁹.12?g·m L^-1?0.47¹4.04?g·mL^-1?0.62¹8.48?g·mL^-1?0.32⁹.45?g·m L^-1,this component has a good linear relationship within the scope.2.Acute toxicity experiment results indicate that,the rats was didn't show any adverse reaction or poisoning symptoms.No deaths occur with maximum dosage of 10 g·kg^-1.3.The dose-effect relationships experiment show that,Rannasangpei pill during 24 h and 48 h could reduce the MCAO rats cerebral infarction rate,and the effective dose was 2.08¹33.36 mg·kg^-1.The intensity of effect increased with the dose in 2.08⁸.33 mg·kg^-1,but in 16.67¹33.36 mg·kg^-1 was not increase with the dose.During cerebral ischemia 24 h,effective dose range was 4.17¹33.36 mg·kg^-1,D_maxax was 33.34 mg·kg^-1,E_max was 3.02%,AUC_tot was 724.22 mg·kg^-1,ED₅₀ was59.33 mg·kg^-1,MRD is 93.96 mg·kg^-1,R was-0.7464;During cerebral ischemia 48h,effective dose range was 2.08¹33.36 mg·kg^-1,D_maxax was 66.68 mg·kg^-1,E_max was2.13%,AUC_tot was 468.54 mg·kg^-1,ED₅₀ was 48.66 mg·kg^-1,MRD is 80.82 mg·kg^-1,R was-0.7458;During cerebral ischemia 48 h,133.36 mg·kg^-1of Rannasangpei pill can improve neurobehavioral abnormalities and inhibit body weight loss on rats.The0.52?1.04?2.08?4.17 and 33.34 mg·kg^-1dose group of Rannasangpei pill can reduce the MDA content in the serum at 24 h,16.67?33.34?66.68 and 133.36mg·kg^-1 dose group of Rannasangpei pill can reduce the MDA content in the serum at 48 h.The8.33?16.67?33.34 and 133.36 mg·kg^-1 dose group of Rannasangpei pill can enhance T-SOD activity in serum of rats in cerebral ischemia 2 h,the 33.34?66.68 and 133.36mg·kg^-1 dose group of Rannasangpei pill can enhance T-SOD activity in serum of rats in cerebral ischemia 24 h,the 1.04?2.08?8.33?16.67?33.34?66.68 and 133.36mg·kg^-1 dose group of Rannasangpei pill can enhance T-SOD activity in serum of rats in cerebral ischemia 48 h.Conclusion:1.Established the methods for determination content of 6 kinds of plant medicine chemical composition by HPLC in Rannasangpei pill.The method is simple,accurate and reproducible,and can be used for the quality control of Rannasangpei pill.2.Rannasangpei pill reducing the cerebral infarction rate of MCAO rats was increased with the dose,and then the maximum effect was achieved.It can improve the rat's neurobehavioral abnormalities and body weight loss.The working mechanism might lies in suppressing the MDA content through improving T-SOD vitality,thus reducing free radical injury of brain tissue.