Chemical Constituents And High Altitude-induced Fatigue Ameliorating Effect Of Tibetan Medicine Pedicularis Kansuensis

Objective1.To separate the chemical components of Tibetan medicine P.Kansuensis.2.To optimize the extraction technology of Tibetan medicine P.Kansuensis.3.To investigate the antioxidant,anti-hypoxia and anti-fatigue active fractions of Tibetanmedicine P.Kansuensis.4.To establish a preparation method for phenylethanoid glycosides with high purity fromTibetan medicine P.Kansuensis.5.To study the ameliorating effect of Ph Gs from Tibetan medicine P.Kansuensis on highaltitude-induced fatigue.Methods1.D101 macroporous adsorptive resin combined with polyamide chromatographic column wasused to prepare total iridoid glycosides and Ph Gs part of Tibetan medicine P.Kansuensis.Thetarget compounds were obtained by means of medium pressure column chromatography.Thechemical structures were elucidated by MS,NMR and DEPT spectroscopy.2.The content of 4 iridoid glycosides and 2 Ph Gs were measured using HPLC method.Basedon the single-factor test,the optimal ethanol technology was investigated using L9?34?orthogonal method.3.Macroporous adsorptive resin combined with polyamide chromatographic column was usedto obtain each part?water extract part,Ph Gs part,iridoid glycosides part,and high polar part?of P.Kansuensis.The antioxidant properties of different fractions of the P.Kansuensisextract was undertaken with VC as positive control;the anti-hypoxic activity of each part wasexamined using three different hypoxia model;the active fractions of P.Kansuensis werescreened with burden swimming test.Contents of LA,glycogen and ATP,the activities of CKin serum,liver and skeletal muscle on mice in different groups were determined.4.The best macroporous resin was selected by static and dynamic adsorption-desorption from01A1,D101,HPD100,AB-8,XAD-6,DM130,DM-301,DM-201,YWD06 B and YWD06 C,with the contents of Verbascoside and Isoverbascoside as index.The purification technologyparameters of total phenylethanoid glycosides from P.Kansuensis were optimized by selectedmacroporous resin.5.72 BALB/C mice were randomly divided into normoxia group?sterile,0.2 m L/20 g?,hypoxiamodel group?sterile water,0.2 m L/20 g?,Rhodiola group?0.5 g/kg?,Ph Gs-L group?0.1 g.g/kg?,Ph Gs-M group?0.3 g/kg?,and Ph Gs-H group?0.5 g/kg?.Mice in HG,Rhodiola,Ph Gs-L,Ph Gs-M,and Ph Gs-H groups were exposed for seven days to a simulated altitude of8000 m in a specially designed animal decompression chamber that reduced the barometricpressure.The normoxic group was kept at a normal atmospheric pressure 1500 m withcontrolled temperature and humidity that were similar to the conditions in the hypoxicchamber.During hypoxic exposure,entered the decompression chamber at the fixed timeevery day,in a simulated altitude of 4000 m plateau environment lavage for 7 d.Theswimming test and specimen collections were conducted at the altitude of 4000 m after lastintragastrically administration.The swimming time was recorded.The blood routine wasdetected.HE staining was employed to observe the morphological changes of the liver andskeletal muscle.Biochemical technique was used to detect the level of glycogen,MDH,SDH,PK,LDH,PA,LA,Na+K+-ATP,Ca2+Mg2+-ATP,BUN,SOD,MDA,and GSH.Results1.Five iridoid glycosides and three phenylethanoid glycosides were isolated and identifiedrespectively as Euphroside?1?,Ixoroside?2?,Plantarenaloside?3?,Boschanaloside?4?,andBosnarol?5?,Crenatoside?6?,Verbascoside?7?,Isoverbascoside?8?.Compound?2?,?3?,?4?,?5?,?6?were obtained from P.Kansuensis for the first time.2.The optimal ethanol technology for iridoid glycosides was 90 min for 1 time with 8 timesamount of 50% ethanol.The optimal ethanol technology for Ph Gs was 90 min each time for 3times with 8 times amount of 50% ethanol P.Kansuensis from Qinghai had higher contentsof Euphroside,Ixoroside,Plantarenaloside,Boschanaloside,Verbascoside,andIsoverbascoside.3.The IC50 values of DPPH,ABTS,OH radical from water extract part,Ph Gs part,and iridoidglycosides part were 210.3,52.1 and 260.3 ?g/m L;196.3,72.1 and 503.9 ?g/m L;87.1,73.1and 95.6 ?g/m L.Ph Gs from P.Kansuensis showed the highest antioxidant activity amongother three parts.Ph Gs can significantly prolong the survival time of atmospheric pressureairtight hypoxia model,sodium nitrite hematologic hypoxia model and simulated 10000 maltitude acute decompression hypoxia endurance model mice?P<0.01 or P<0.05?.Ph Gsfraction could significantly prolonged the burden swimming time of mice?P<0.01?,decreasethe content of LA,increase the content of glycogen and ATP,and increase the activity of CK?P<0.01 or P<0.05?.4.The best purifying technology conditions of Ph Gs from P.Kansuensis were as follows: theoptimum type resin was D101,the concentration of sample was 20 mg/m L,the flow rate ofsample was 3 BV/h,then wash off the impurity with 3 BV deionized water and eluted by 5BV 50% ethanol with the desorption rate at 2 BV/h.The 50% ethanol fraction wasevaporated and dried with cry desiccation to obtain the total Ph Gs.The contents ofVerbascoside and Isoverbascoside in total Ph Gs were 42.29% and 28.51%,respectively.5.Ph Gs from P.Kansuensis can significantly prolong the survival time of atmospheric pressureairtight hypoxia model,protect the liver and skeletal muscle in mice from damage,reduce theRBC,HCT,WBC,HGB,MCH,MCHC,decrease the contents of LA,BUN,MDA,increasethe contents of PA,GSH,glycogen,and improve the activity of MDH,SDH,PK,Na⁺K⁺-ATPase,Ca⁺²Mg⁺²-ATPase,and SOD,and induce decrease of LDH activity?P<0.01or P<0.05?.Conclusion1.The method exhibits better performance of separation,which is simple and practicable,canbe used for the qualitative analysis and rapid preparation of 5 iridoid glycosides and 3 Ph Gsfrom Tibetan medicine P.Kansuensis.2.This extraction method is reasonable,stable,and feasible.The content of iridoid glycosidesand Ph Gs from different habitats has some differences.3.Ph Gs of P.Kansuensis possess potent antioxidant and anti-hypoxia activity,which is a naturalantioxidant and anti-hypoxia supplement.Ph Gs fraction may be the anti-fatigue activeingredient of P.Kansuensis.4.This method can be used for the large-scale preparation of Ph Gs with high purity,which israpid,simple and practicable with high efficiency.5.Ph Gs of P.Kansuensis can ameliorate high altitude-induced fatigue by stabling the peripheralhemogram index,maintaining the normal physiological structure,improving the utilizationrate of energy storage material,avoiding energy metabolism disorders,and enhancing thecapacity of oxidative stress.