Active Constituents From Traditional Chinese Medicine Against Acute Lymphoblastic Leukemia

Acute lymphoblastic leukemia(ALL) is a malignant(clonal) disease of the bone marrow in which early lymphoid precursors proliferate and replace the normal hematopoietic cells of the marrow.ALL may be distinguished from other malignant lymphoid disorders by the immunophenotype of the cells,which is similar to B-or T-precursor cells.Chemotherapy,which uses powerful drugs,including alkylating agents, anti-metabolism drugs,antibiotics and hormones,to destroy cancer cells,has often been clinically used to treat leukemia.Although chemotherapy can achieve a high five-year disease-free survival rate,it also leads to strong and serious side effects,consequently, affecting the patients' quality of life,in particular for those children patients.Traditional Chinese medicine(TCM) has been widely used for the treatment of leukemia in China with less side effects observed in clinical.However,the active ingredients in those traditional Chinese medicines are largely unknown and uninvestigated.Therefore,the main objectives of this thesis were to elucidate the active compounds responsible for the pharmacological activity of those Chinese medicines against ALL and to identify new potential lead compounds for the anti-ALL drug discovery.In this thesis,24 species of the most frequently used traditional Chinese medicines for the treatment of leukemia were selected,and the bioactivities of their alcohol extracts against three different leukemia cell lines(HL-60,REH and Raji) were screened by MTT assay in vitro.The preliminary results showed that the extracts of Gardenia jasminoides, Dryopteris crassirhizoma and Scutellaria barbarta effectively inhibited proliferation of REH cells,a human B cell line,indicating the potential activity against ALL.The constituents from the active fractions of G.jasminoides against ALL were isolated by various column chromatographic techniques,leading to the purification of 8 compounds.Their structures were elucidated on the basis of spectral analysis(IR, ESI-MS,HRMS,1H-NMR,13C-NMR,HMBC,HMQC),and identified asβ-sitosterol (ZZ1),5-hydroxy-7,3',4',5'-tetramethoxyflavone(ZZ2),ursolic acid(ZZ3),D-mannitol (ZZ4),geniposide(ZZ5),deacetylasperulosidic acid methyl ester(ZZ6),genipin-1-β-gen -tiobioside(ZZ7) and scandoside methyl ester(ZZ8),respectively.Results showed that compound ZZ3 exhibited inhibitory effects on REH cell proliferation in vitro with IC50 being 7.5μg·mL-1.Ursolic acid might be one of the active ingredients responsible for the effect of anti-acute lymphoblastic leukemia conferred by G.jasminoides. Nine compounds were isolated from the CH₂Cl₂ fraction of the ethanol extract of D. crassirhizoma,which were found to inhibit the proliferation of REH cells.On the basis of the spectroscopic analysis and chemical methods,their structures were determined as pentacosanol(GZ1),3-Methyl-butyrylphloroglucinol(GZ3),3-hydroxy-heptacosanol (GZ4),flavaspidic acid AB(GZ5),dibutylphthalate(GZ6),di-isobutylphthalate(GZ7),β-sitosterol(GZ8) and norflavaspidic acid AB(GZ9).Of the nine compounds isolated, GZ6 and GZ7 were isolated from this genus for the first time,GZ3 and GZ9 from this plant for the first time.The MTT assay showed that GZ3 showed strong inhibition activity against REH cells with an IC₅₀ of 8.0μg·mL^-1,GZ5 and GZ9 displayed IC₅₀ of 35.3μg·mL^-1 and 32.2μg·mL^-1 against the same cell line.3-Methyl-butyrylphloroglucinol might be the active compound from D.crassirhizoma agaist acute lymphoblastic leukemiaEleven compounds were obtained from the active fraction,the ethyl acetate parts of the alcoholic extract of S.barbarta,and identified as 3-hydroxy-heptacosanol(BZL3), 4'-hydroxywogonin(BZL4),chlorogenic acid(BZL5),oroxylin-A(BZL6),naringenin (BZL7),wogonin(BZL8),baicalein(BZL9),daucosterol(BZL10),β-sitosterol(BZL11), quercetin(BZL12) and baicalin(BZL13),respectively.Amongst of the compounds, BZL3 and BZL6 were isolated from this plant for the first time.BZL4 was found to show inhibitory activity against REH cells with an IC₅₀ of 13.9μg·mL^-1.In addition,BZL5 and BZL7 appeared also to inhibit the proliferation of REH cells,but to a lesser extent,and show IC₅₀ of 40.1μg·mL^-1 and 43.0μg·mL^-1,respectively.4'-hydroxywogonin may be responsible for anti-acute lymphoblastic leukemia.