Extraction And Enrichment Of Total Triterpenes From Of Sibiraea Angustata (Rehd.) Hand.-Mazz.on Its Anti-hypoxia Activity

In this paper,total triterpenoids in Sibraea angusta（Rehd.）Hand.Mazz（SATT）were studied as the object.First of all,Optimizate the extraction and enrichment process from Sibraea angusta（Rehd.）Hand.Mazz.Then studied the best hypoxia injury Vehicle.Finally,we studied the protective effects of SATT on hypoxia rats.First:Research on the extraction and enrichment process of SATT.Select 70%ethanol extracts as the effective extracts by anti-hypoxia activities.Through L_{9（）3⁴}orthogonal test,we decide the final extraction process was using 70%ethanol,extracted 2 times,1.5h each time,the SATT content was up to 32.25%.The best enrichment process was AB-8 type resin.The sample concentration was 2 mg/ml,eluted with 80%ethanol of 5 BV（Bed Volume）,and the final SATT content was up to 61.09%.Second,:Establishment of rats Vehicle of hypoxic injury at high altitude.Male Wistar rats were randomly divided into Control group,4000 m group,6000 m group,and 8000 m group.Rats in each group were hypoxic for 3 days.Rats’heart rate（HR）and diastolic blood pressure（DBP）,Systolic blood pressure（SBP）,mean arterial pressure（MBP）were measured during hypoxia.Then measured the blood gas and blood routine of rats.After the end of hypoxia,the rats were sacrificed,and the MDA,T-SOD,NO,LD,LDH,Na⁺-K⁺-ATPase,and Ca²⁺-Mg²⁺-ATPase in the myocardium and brain were measured.HE staining was used to observe the structural changes of myocardium and brain tissue.Compared with the Control group,the HR of hypoxic rats increased significantly,SBP,DBP,MBP decreased significantly,arterial blood pO₂,pCO₂,SO₂ decreased significantly（P<0.05 or P<0.01）,WBC,RBC,PLT,PCT,Hb,and Hct increased significantly（P<0.05 or P<0.01）.The contents of MDA,LD,LDH,and NO in the myocardium and brain tissue were significantly increased（P<0.01 or P<0.05）,reaching the maximum at a simulated hypoxia of 8000 m 3 days（P<0.05 orP<0.01）,and the activities of T-SOD,Na⁺-K⁺-ATPase,and Ca²⁺-Mg²⁺-ATPase weresignificantly decreased,reaching the minum at a simulated hypoxia of 8000 m 3 days（P<0.05or P<0.01）.The results of HE staining showed that the myocardial and brain tissues of rats in each hypoxia group were significantly damaged,and the damage was most severe in hypoxia at 3 days in simulated altitude 8000 m group.Third,The study on anti-hypoxic activity of SATT.60 male Wister rats were implanted into telemeters and randomly divided into Control group,Vehicle group,Acetazolamide group（ACZ）,and low dose（SATT-L）,middle dose（SATT-M）,high dose（SATT-H）.Rats in the administration group were given intragastric administration for 7 days and hypoxia for 3 days.The heart rate（HR）,diastolic blood pressure（DBP）,systolic blood pressure（SBP）,and mean arterial pressure（MBP）were measured during hypoxia and then the blood gas and blood routine of the rats were measured.HE staining and transmission electron microscopy were used to observe the changes of heart and brain structure in rats.After the heart and brain were homogenized,their biochemical parameters were determined,including MDA,T-SOD,NO,LD,LDH,Na⁺-K⁺-ATPase,and Ca²⁺-Mg²⁺-ATPase.The expression of HIF-1a,Nrf 2（nuclear and cytoplasmic）,Caspase-3,GCLC,HO-1,Bax,and Bcl-2 in myocardium and brain tissue and the autophagy-related protein Mfn 2 in myocardial mitochondria were observed by Western blot expression.The experimental results showed that comparing with the vehicle group,SATT can significantly decrease the HR,improve SBP,DBP and MBP in rats,and increase the pO₂,pCO₂,and SO₂ in the blood（P<0.05 or P<0.01）,reduce WBC,RBC,PLT,PCT,Hb（P<0.05 or P<0.01）.Simultaneously,SATT can decrease the contents of MDA,LD,and NO in myocardium and brain tissues（P<0.05 or P<0.01）,and increase T-SOD,Na⁺-K⁺-ATPase,and Ca²⁺-Mg²⁺-ATPase activities（P<0.05 or P<0.01）in rets.The results of Western-Blot showed that SATT can down-regulate the expression of HIF-1a and VEGF in the heart and brain of hypoxic rats（P<0.05）,regulate vascular permeability,and promote vascular remodeling.Meanwhile,SATT can down-regulate Nrf 2,Caspase-3,GCLC,HO-1protein expression（P<0.05）,remove excess free radicals and ROS in vivo;SATT can down-regulate Bax,up-regulate the expression of Bcl-2 protein to inhibit cell apoptosis（P<0.05）.It showed that SATT can regulate the body’s energy metabolism and inhibit cell apoptosis to achieve the protective effects of high altitude hypoxic rats.