Studies On The Extraction,Separation,Antitumor Activity And Derivatization Of The Diterpenoids From Isodon Japonica Var.Glaucocalyx

Background:Isodon japonica var.glaucocalyx,which belongs to the genus Isodon and the family Labiatae,commonly used as antiinflammatory,antitumor,antiarthralgia and snake bites drug in traditional Chinese medicine.Research shows that Isodon japonica var.glaucocalyx is rich of ent-kaurane diterpenoids with antitumor activity.Object:This thesis focuses on the investigate of the chemical constituents and the antitumor activity of Isodon japonica var.glaucocalyx,collected from Huixian,Henan Province,and providing support for further investigation.In addition,providing crude material for structure qualification of glaucocalyxin A（GLA）.To synthesize a series of GLA derivations by structure qualification,in order to enhance the antitumor activity and obtain the effect of targeted therapy.Method:The chemical constituents were isolated and purified by means of repeated silica column chromatography and recrystallization from the Ethyl Acrylate portion of70%acetone extract of the leaves of Isodon japonica var.glaucocalyx.Their structures were identified by spectral analysis and the antitumor activity of seven diterpenoids was evaluated by MTT assay.The effects of compound 1 on morphological changes of tumor cells was observed under reverse fluorescence microscopy.Starting from GLA without altering the pharmacophore?,β-methylene cyclopentanone,dicarboxylic acid derivatives and amino acid derivatives with better water-solubility were sythesized through structure modification of 7,14-dihydroxy.Moreover,GLA-sigma-2 receptor ligand conjugates were sythesized by linking with dicarboxylic acid derivatives.The structures of all compounds were characterized by morden spectra analysis including ¹H-NMR,¹³C-NMR and HR-ESI-MS.Result:Twelve known compounds were identified from the leaves of Isodon japonica vat.glaucocalyx.Seven of them were ent-kaurane diterpenoids.The antitumor activities of the seven diterpenoids were evaluated by MTT assay.The result indicated that compounds 1（GLA）,2 and 3 shown the potent activity in vitro against Hep G2 and Hela tumor cell lines with IC₅₀ values less than 10μM,among which GLA against Hep G2 with IC₅₀ values 3.94μM.Apoptotic cells were observed in Hep G2 cells treated with compound 1 under reverse fluorescence microscopy.Sixteen GLA derivatives were sythesized,including four dicarboxylic acid derivatives eight GLA-sigma-2 receptor ligand conjugates and four amino acid derivatives.Conclusion:The chemical constituents of Isodon japonica var.glaucocalyx,collected from Huixian,Henan Province are basically the same comparing with that of Linzhou,Henan Province.The 100g pure GLA will as the materials of structure modification.The structure modification will open the synthetic route of dicarboxylic acid derivatives and amino acid derivatives of GLA 7,14-dihydroxy as well as GLA-sigma-2 receptor ligand conjugates.