| The dried rhizome of Trillium tschonoskii Maxim.(RTT,Tou ding yi ke zhu in Chinese),which belongs to genus Trillium(Liliaceae),is used as a folk medicine in China.In this study,the chemical constituents of the RTT were analyzed using ultra-high-performance liquid chromatography coupled with linear ion trap and orbitrap high resolution mass spectrometry(UPLC-LTQ-Orbitrap-MS)methods.An efficient HPLC method for simultaneous determination of three main steroidal saponins in the herbal material was established.The total steroidal saponins of T.tschonoskii(TSTT)was prepared and its main chemical constituents were isolated and identificated.Furthermore,the techniques of neural behavior,MRI,neuropathology were applied to evaluate the anti-cerebral ischemia effects of TSTT.Then,the study focused on the molecular mechanism of TSTT to promote neurogenesis after ischemia through Wnt/?-catenin signaling pathway.1 Chemical study of the rhizome of T.tschonoskii Objective:To identificate and determine the main chemical constituents in the rhizome of T.tschonoskii.Method:The main components of RTT were systematically studied with UPLC-LTQ-Orbitrap-MS method.Three steroid saponins,including Paris saponin VI,Pennogenin-3-O-?-L-rhamnopyranosyl(1?4)-[O-?-L-rhamnopyranosyl(1?2)]-O-?-D-glucopyranoside(PRRG)and Paris saponin VII in RTT were determined using HPLC-DAD method.The assay was achieved on an Agilent Poroshell C18 column(100 mmx4.6 mm,2.7 ?m)at 30?,using a gradient mobile phase consisting of acetonitrile and water.The flow rate of was 1.0 mLˇmin-1 and the wavelength was set at 203 nm.RTT was extracted with 70%ethanol.The extraction was partitioned with n-butanol and then isolated by D101 macroporous resin to yield the total steroid saponins of T.tschonoskii.The main constituents in TSTT were chromatographically isolated and identificated by spectroscopic methods.In addition,the qualitative analysis was conducted with UHPLC-LTQ-Orbitrap-MS methods.A systematic strategy,including mass database hitting and high accurate MSIMS information was used.Results:27 chemical constituents were identified form RTT,including 23 steroid saponins.The determination result showed that HPLC peaks for Paris saponin ?,Paris saponin ? and PRRG had good separation.The correlation of standard curve met requirement in the linear range(r>0.9997);the average recovery was 96%?103%with RSD from 0.57%to 3.61%.The content of 3 constituents in 11 batches of RTT had obvious differences.Four saponins were isolated from TSTT and identificated as follows:paris saponin ?.Pennogenin-3-O-?-L-rhamnopyranosyl-(1 ? 4)-[0-?-L-rhamnopyranosyl-(1?2)]-0-?-D-glucopyranoside(PRRG),Paris saponin ? and Pennogenin-3-O-?-D-glucopyranoside.Seven steroidal saponins in TSTT was identificated by LC-MS technology,including Paris saponin ?(1),PRRG(2),Paris saponin ?(3),Pennogenin-3-O-a-L-rhamnopyranosyl-(1?4)-[O-?-L-rhamnopyranosyl-(1? 4)]-O-?-D-glucopyranoside(4),Pennogenin-3-O-?-L-rhamnopyranosyl-(1?4)-O-?-D-glucopyranoside(5),Pennogenin-3-O-?-D-glucopyranoside(6),Paris saponin ?(7).Conclusion:A simple and effective method was developed for characterization of steroid constituents in the rhizome of T.tschonoskii with UPLC-LTQ-Orbitrap-MS method.The main steroid saponins in RTT were isdentificated.The HPLC determination method was simple and effective and can be used for quality control of RTT or its extract.The principle constituents of TSTT included six pennogenyl-type and one diosgenyl-type saponins.These results provided essential data for further pharmacodynamics research.2 Study of Trillium tschonoskii Maxim.on anti-cerebral ischemia pharmacological activities:Objective:To investigate the effects of TSTT on neural behavior,tissue injury,neuropathology,cerebral ultrastructure of permanent Middle Cerebral Artery Occlusion(pMACO)rats.Then,the study focused on the molecular mechanism of TSTT to promote neurogenesis after ischemia through Wnt/?-catenin signaling pathway.Methods:The study adopted the animal model of permanent Middle Cerebral Artery Occlusion(pMACO).Sprague-Dawley rats were randomly divided into sham operation group,model group,TSTT 130 mg/kg group,TSTT 65 mg/kg group,TSTT 33 mg/kg group and EGB 761 60 mg/kg group.TSTT was orally administered 6 h after pMACO and daily for the next successive 15 days,sham operation group and model group rats were administered with normal saline at the same volume.Bederson neurologic score was performed daily and beam walking test was conducted to investigate the influence of TSTT on motor function of cerebral ischemic 15d rats.T2-weighted imaging(T2WI)were performed to evaluate change of cerebral infarct volume and T2 mapping were used to quantitatively evaluate different encephalic regions injury degree of TSTT;Three dimensional time of flight Magnetic resonance angiography(3D-TOF MRA)images were used to observe the degree of middle cerebral artery occlusion and quantitatively analyze the changes in vascular signal intensity after stroke.Three dimensional arterial spin labeling(3D-ASL)provided the information of cerebral blood flow(CBF)after pMCAO and effects of TSTT on haemodynamics of focal cerebral ischemia rats were quantitatively analyzed.The morphologies change of neurons after focal cerebral ischemia in grey and white matter was observed with HE staining;The expression of APP,cell proliferation antigen marker Ki67,neuronal migration marker protein DCX,GFAP+/Ki67+ and NeuN+/Ki67+ positive cells were observed by immunofluorescence staining to comprehensively assess the impacts of TSTT on MCAO rats'neuropathology.The ultrastructure of neurovascular unit,axon and myelin sheath,synapsis and mitochondria was observed by transmission electron microscopy to estimate the protection function of TSTT on ischemic rats.The mRNA expression of Wnt3a??-catenin?GSK-?/??Dishevelle and CRMP 2 were observed by RT-PCR;The protein expression of Wnt3a?Dishevelled 3,?-catenin and P-?-catenin Ser33/37 Thr41,GSK-3 ? and P-GSK3 ? Ser21/Tyr279,GSK-3? and P-GSK3? Ser9/Tyr216,CRMP 2 and P-CRMP2 Thr514 were observed by Western Blotting.Result:Kaplan-Meier estimates revealed that the survival rate significantly increased in TSTT 65 mg/kg rats compared with the model group(P<0.05);TSTT 65 mg/kg could improve cerebral ischemia rats neurobehavioral defect(P<0.01 or P<0.05),reduce infarct volume(P<0.01),decrease rT2 values of of the ipsilateral parietal cortex and striatum(P<0.05)and improve signal of blood vessel and cerebral blood flow of cortex and striatum(P<0.01 or P<0.05).The result of HE staining showed lightened grey and white matter injury in TSTT 65 mg/kg group rats.The expression of APP protein in grey matter:cortex,thalamus,striatum and white matter:external capsule,internal caosule were decreased in TSTT 65 mg/kg group rats compared with the model group(p<0.01).The expression of Ki67 in SVZ,striatum region and the expression of DCX in striatum were increased in TSTT 65 mg/kg group rats compared with the model group(P<0.01).TEM results revealed that TSTT 130,65,33 mg/kg could protect NVU and the recovery degree of injured neurons,blood vessels and astrocytes of TSTT 65 mg/kg group rats was more better than that of TSTT 130,33 mg/kg group rats;the ratio of anxon external diameter/inner diameter significantly decreased in TSTT 130?65?33 mg/kg group rats;The numbers of synapsis were increased,the length,depth of PSD-95 were grown and synaptic cleft was decreased in TSTT 130,65 mg/kg group rats compared with the model group(P<0.01),for TSTT 33 mg/kg group,the numbers of synapsis were increased,the depth of PSD-95 were grown and synaptic cleft was decreased(P<0.01);the mitochondria structure of TSTT 130.65.33 mg/kg group rats were basically remained intact and the mitochondrial damage score decreased as compared to the model group rats(P<0.01).RT-PCR anlysis results demonstrated that the mRNA expression of Wnt3a in TSTT 65 mg/kg rats was up regulated(P<0.01);TSTT 130,65 mg/kg could up regulated ?-catenin mRNA expression(P<0.05),TSTT 65?33 mg/kg could increase Dishevelled 3 mRNA expression compared to model group(P<0.05);TSTT 65?33 could down regulate GSK-3? mRNA,GSK-3? mRNA expression(P<0.05),TSTT 130 mg/kg could down regulate GSK-3? mRNA expression(P<0.01);TSTT 65 mg/kg could increase CRMP 2 mRNA expression(P<0.05).Western Blot anlysis results demonstrated that the protein expression of Wnt3a in TSTT 65?33 mg/kg rats was up regulated(P ? 0.05);TSTT 65 mg/kg could up regulated ?-catenin expression(P<0.05),TSTT 130,65,33 mg/kg could decrease P-?-catenin Ser33/37 Thr41 expression(P<0.05)and increase Dishevelled 3 protein expression compared to model group(P<0.01 or P<0.05);TSTT 65mg/kg could increase P-GSK3?/? Ser21/9 expression(P<0.05),TSTT 65?33 mg/kg could increase CRMP 2 protein expression(P<0.05)and decrease PCRMP2 Thr514 expression(P<0.01);TSTT 130 mg/kg could decrease PCRMP2 Thr514 expression(P<0.01)compared to the model group.Conclusion:TSTT can reduce focal cerebral ischemia rats tissue damage caused by middle cerebral artery occlusion,improve neuropathology and NVU microstructure changes,increase ischemic brain blood flow;TSTT can promote neurogenesis and reshape neural function after cerebral ischemia injury through regulating key signaling molecules transcription and translation in Wnt/?-catenin pathway. |
PDF Full Text Request