Anti-fatigue Activity Of The Mixture Of Cistanche Phenylethanol Glycosides And Gardenia Yellow Pigment In Hypoxia

Objective1.Screen the active ingredients of anti-hypoxia and anti-fatigue in Gardenia.2.Optimize the best proportion of Cpg and Gyp mixture on anti-hypoxia activity using the normal pressure airtight test.3.Evaluate the anti-fatigue effect of the optimized Cpg and Gyp mixture under the normoxia condition.4.Evaluate the anti-fatigue effect of the optimized Cpg and Gyp mixture under the hypoxic condition.5.Prepare the granules of Cpg and Gyp.Methods1.Four constituents（Gardenia water extract,Gardenia yellow pigment,Gardenia polysaccharide and Gardenia iridoid glycosides）in Gardenia were obtained with chromatograph columns.The anti-hypoxia activity constituent was screened by determining the survival time of mice in hypoxia.And the safety of each constituent in Gardenia was compared by preliminary toxicity evaluation in mice.The anti-hypoxia effects of three doses of Gyp were evaluated with the survival time and the water content of lung tissue and brain tissue in atmospheric closed experiment.The hypoxia model of mice was established by the hypobaric hypoxia experimental cabin,and the effect of Gyp on the exercise fatigue in hypoxia was studied through exhaustive swimming test.2.The proportion of Cpg and Gyp mixture with the best anti-hypoxia activity was optimized by normal atmospheric airtight experiment.And the anti-hypoxia effect of the optimized mixture was further studied with atmospheric pressure airtight hypoxia model,sodium nitrite hematologic hypoxia model,and acute hypoxia model.3.In the further study,50 BALB/C mice were randomly divided into Control group（sterilized water,0.1 ml/10 g）;Positive group(Rhodiola capsule,0.50 g·kg^-1·d^-1),Drug groups(Cpg and Gyp 1:1 mixture;Drug-L,0.10 g·kg^-1·d^-1;Drug-M,0.30 g·kg^-1·d^-1;Drug-H,0.50g·kg^-1·d^-1).All mice were continuous administrated for 7 days,and 1 h after the last administration in the seventh day;mice were put in a pool to swim with burden（7%body weight）.The exhausted swimming time of every mouse was recorded;the level of BUN,PA,LD,LDH,GSH,T-SOD,MDH,SDH,Na⁺-K⁺-ATPase,Ga²⁺-Mg²⁺-ATPase,HG,and MG in tissues were detected;the protein expression levels of Bax,bcl-2 and caspase-3 in tissues were detected by WB.4.As to the study on the anti-fatigue effect in hypoxia,75 Wistar rats were randomly divided into 5 groups:Normal oxygen blank control group（NC,distilled water,0.1 ml/10 g）,Hypoxia blank control group（HC,distilled water,0.1 ml/10 g）,Exhausted swimming blank group（EC,distilled water,0.1 ml/10 g）,Exhausted swimming positive control group(EP,rhodiola capsule,0.35 g·kg^-1·d^-1),Exhausted swimming drug group(ED,Cpg and Gyp mixture（1:1）,0.35g·kg^-1·d^-1).Except the NC group,the other rats were placed in the experimental module with a simulating altitude of 8000 m,and were given the drug in the simulated 3500 m altitude environment for five days.After the last administration,the rats of EC,EP and ED groups were carried out burden swimming test in an altitude of 3500 m,and the time of exhausted swimming was recorded.The pathological changes of brain,liver and skeletal muscle tissue were observed by HE staining.The level of BUN,UA,CRE,CK,LD,LDH,PA,MDA,T-SOD,NOS,NO,CAT,GSH-PX,ATP,HG and MG in the serum or tissues were determined by biochemical method.The expression levels of Bax,bcl-2,Nox2 and Ampk proteins were detected by WB.5.Starch,dextrin,and lactose were selected as auxiliary materials to prepare granules of Cpg and Gyp mixture（1:1）with 75%ethanol as binder.According to the stipulation of the related indexes in the Pharmacopoeia,the prepared granules were evaluated,and the proportion of auxiliary materials was screened.The quality for the final granules was estimated accoriding the Pharmacopoeia.Results1.In the four constituents of Gardenia,Gyp showed significant anti-hypoxia effect with a dose depending relationship,and the Gyp is safe;Compared with the control group,Gyp-H group was able to prolong the anti-hypoxia time of the mice（P<0.05）.Gyp can reduce the water content in brain and lung tissue（P<0.05,0.01）.Gyp can prolong the exhausted time of burden swimming mice in hypoxia environment.Compared with model group,Gyp-M and Gyp-H could increase exhausted swimming time significantly（P<0.05 or P<0.01）.2.The mixture of Cpg and Gyp showed anti-hypoxia potential.The anti-hypoxia activity of 1:1 mixture was optimization,and could prolong the mice survival time in hypoxic,reduced the mortality in acute hypoxia mice significantly（P<0.05）.3.Compared with the control group,each dose group of the optimized Cpg and Gyp mixture could prolong the exhausted swimming time of mice.And the exhausted swimming time in Drug-L and Drug-H group were prolonged significantly（P<0.05）.Compared with the control group,the optimized mixture can reduce the content of BUN,increase PA,GSH,HG and MG,enhance the activity of T-SOD,LD,SDH and LDH,MDH,Na⁺-K⁺-ATPase and Ga²⁺-Mg²⁺-ATPase.Compared with control group,each dose groups can decrease the Caspase-3protein expression levels in liver and brain tissue（P<0.05）.And Drug-L and Drug-H can significantly reduce the ratio of Bax and Bcl-2 in mice liver and brain tissue（P<0.05）.4.Compared with the EC group,exhausted swimming time of the EP group and the ED group was prolonged significantly（P<0.05）.The Cpg and Gyp 1:1 mixture could reduce the content of BUN,UA and CRE in serum,decrease the content of PA,CK,MDA,NOS and NO in serum or tissues,increase the level of CAT,GSH-PX,T-SOD,LD,LDH,ATP,HG and MG.It also could reduce the expression level of Nox2 and Ampk and the ratio of Bax/bcl-2,and reduce the infiltration of inflammatory cell,ameliorate the histopathological changes.5.The best prescription of the granules was selected as lactose:drug=2:1.And the verification test showed that the granules have better granularity,dissolubility,formability and stability.Conclusion1.The anti-hypoxia and anti-fatigue effective ingredient of gardenia is Gyp,and the Gyp is safe.2.The mixture of Cpg and Gyp has good anti-hypoxia effect and the optimal proportion is1:1.3.The optimized mixture could prolong the swimming time of mice significantly,and relieved the exercise fatigue.4.The optimized mixture of Cpg and Gyp could prolong the swimming time of rats in the simulated plateau condition,enhance the elimination process for metabolites in vivo,increase energy storage,increase the scavenging activity for free radical,increase the activity of related enzymes,decrease the expression of apoptotic protein,and inhibit inflammatory cells.5.The preparation method of Cistanche phenylethanoid glycosides and Gardenia yellow pigment mixture granules was stable and feasible.