Chemical Constituents From Rhizome Of Typhonium Giganteum Engl.and Their In Vitro Neuroprotective Activities

Typhonii Rhizoma is the dried rhizome of Typhonium giganteum Engl.,which belongs to the Araceae family of the Typhonium genus.As a traditional Chinese medicine,it has the effects of "dispelling wind-phlegm" and is utilized to treat stroke,epilepsia,tetanus,migrane and so on.Up to now,the limited phytochemical investigations on T.giganteum have led to the presences of fatty acids,lignans,cerebrosides,nucleosides and hydantoins.Moreover,modern pharmacological researches have demonstrated that the ethanol extract from Rhizoma Typhonii showed significant neuroprotection against ischemic brain damage.However,few of studies on its active constituents are reported.Herein,this paper aims to figure out the relationship between chemical components from Typhonii Rhizoma and their neuroprotective activities.The anticipate results of this study will establish a foundation for the finding of active constituents from Rhizoma Typhonii and research&development of new medicines.The main research contents are summarized as fol ows:In chapter 1,we focused our studies on chemical constituents from Typhonii Rhizoma.70%EtOH extract form Rhizoma Typhonii was first suspended in water and successively partitioned with petroleum ether,ethyl acetate and n-butanol,accordingly to give three portions.Then 60 compounds were isolated from ethyl acetate section and n-butanol section by silica gel,ODS,Sephadex LH-20 column chromatography and HPLC,respectively.Finally,the structures of 44 compounds were fully identified by the physicochemical determination and spectral analysis(IR,UV,MS and NMR).Among them,there are 13 cerebrosides,8 lipids,4 steroids,18 N-heterocycle compounds and 1 amino acid.To the best of our knowledge,compounds 16 and 17 are two new compounds,which were separately named as typhonoside E(1-O-?-D-glucopyranosyl-(2S,3R,4E,8E)-2-[2'(R)-hydroxyhenicosano-yl-amino]-4,8-octadecadiene-1,3-diol)and typhonoside F(1-O-?-D-glucopyranosyl-(2S,3R,4E,8Z)-2-[2'(R)-hydroxyhenicosanoyl-amino]-4,8-octadecadiene-1,3-diol).Compounds 24,25,43 and 44 were obtained for the first time from the family Araceae.Compounds 20,21,30 were found from the genus for the first time.Compounds 1,4,5,7,8,27,28 and 36 were isolated from the plant for the first time.In particular,compounds 43 and 44(two berberine alkaloids)were separated from the Monocotyledoneae(Araceae family)for the first time.In chapter 2,we mainly investigated in vitro neuroprotective activities of cerebroside compounds 10-22.First,MTT assay was used to determine the concentration of glutamate for PC 12 cells injury.The results showed that the survival rate of PC 12 cells was nearly 50%after treated with 12.5 mmol/L glutamate for 24 h.Then,the neuroprotective activities of cerebroside compounds 10-22 were screened.As expected,all the cerebroside compounds 10-22 could protect PC 12 cells from glutamate injury.The structure-activity relationship analysis indicated that the neuroprotective activities of cerebroside compounds were correlated with the length of fatty acyl chain.In chapter 3,we focused on the cytoprotection and mechanism of compound 20 on PC 12 cells injury induced by glutamate.We first tested the protective effects of compound 20 on PC 12 cells by morphology and viability,and the anti-apoptosis effects by flow cytometry.The results of MTT assay showed that the protective effects of compound 20 against glutamate-induced injury in PC 12 cells were concentration-dependent.The results of Annexin V-FITC/PI staining showed that compound 20 could decrease cell apoptosis induced by glutamate.To further explore the neuroprotective mechanism of compound 20,we performed Western blot.The results exhibited that,comparing with glutamate model group,compound 20 treated groups could significantly down-regulate expression of Bax protein,up-regulate expression of Bcl-2 protein and could also reduce the expression of Caspase-3 protein induced by glutamate.