Study On Isolation And Identification Of Chemical Constituents And Anti-tumor Activity Of Alocasia Macrorrhiza

ObjectiveAlocasia macrorrhiza（Linn.）Schott belongs to Alocasia genus of Araceae family.It has the functions of clearing away heat and toxic toxicity,detoxifying swelling and dissipating stagnation.It is also used to treat tumors in the folk.At present,there are few studies on its chemical composition and related anti-tumor activity.In this paper,MTT experiment was used to guide the tracking and separation of active ingredients,and plant chemistry was used to study its internal chemical components,providing certain experimental basis for the clinical research and application of Alocasia macrorrhiza（Linn.）Schott.Methods（1）Anti-tumor activity screening of different fractionAlcoholic extract obtained from the dried herb of Alocasia macrorrhiza was divided into five portions with the solvent of petroleum ether,dichloromethane,ethyl acetate,n-butyl alcohol and water.The anti-tumor activity of the fractions was evaluated with MTT assay on two kinds of tumor cell lines（prostate cancer cells PC3,Mouse melanoma cells B16）to screen the active fractions.（2）Isolation and identification of chemical constituentsThe active fractions were separated and purified by column chromatography,recrystallization and other separation and purification methods.The monomeric compounds were identified by chemical and spectral analysis(¹H-NMR,¹³C-NMR,MS,et al).（3）Anti-tumor activity screening of chemical compoundsFinally the monosomic compounds were screened by cell assays（means of MTT）in vitro to find out the anti-tumor active ingredients of Alocasia macrorrhiza.（4）Preliminary study on anti-tumor effect of compound 1The anti-tumor activity was determined by means of MTT method and Hochest 33342staining method.Results（1）Anti-tumor activity screening of different fractionThe results of cell assays in vitro was displayed that the fraction of petroleum ether,dichloromethane and ethyl acetate showed definite anti-tumor activity.Their IC₅₀values were48.97μg/m L,61.81μg/m L,449.80μg/m L against PC3,and 218.20μg/m L,213.00μg/m L,237.90μg/m L against B16.The anti-tumor activity of petroleum ether was the best,which was more sensitive to PC3 cells.（2）Isolation and identification of chemical constituentsThe petroleum ether fraction was isolated and purified systematically,and 5 compounds were successfully isolated and identified,including Palmitic acid（1）,Stigmasterol（2）,Epicampesterol（3）,β-sitosterol（4）,Tri（2,4-di-tert-butylphenyl）phosphate（5）.（3）Anti-tumor activity screening of chemical compounds3 active ingredients were found by screening above chemical compounds.The IC₅₀values of compound 1 of PC3 and B16 were 37.14μg/m L and 51.20μg/m L.The IC₅₀values of compound 3 of PC3 and B16 were 133.70μg/m L and 51.90μg/m L.The IC₅₀values of compound 4 of B16 was 194.10μg/m L.（4）Preliminary study on anti-tumor activity of compound 1Compound 1 could inhibit PC3 and B16.The IC₅₀values of compound 1of PC3 were67.35μg/m L,37.14μg/m L and 29.71μg/m L at 24h,48h and 72h.The IC₅₀values of compound1 of B16 were 118.9μg/m L,51.20μg/m L and 33.02μg/m L at 24h,48h and 72h.Hochest33342 staining method showed that,after the intervention of compound 1 on PC3 cells and B16 cells,part of the cell nucleus concentrated,crescent-shaped or fragmented into a plurality of spherical particles.And with the increase of the concentration and the extension of time,The cells showed apoptosis phenomenon with different degrees.ConclusionPetroleum ethyl fraction which was more sensitive to PC3 cells have the strongest anti-tumor activity.Five compounds were obtained from Alocasia macrorrhiza,including Palmitic acid（1）,Stigmasterol（2）,Epicampesterol（3）,β-sitosterol（4）,Tri（2,4-di-tert-butylphenyl）phosphate（5）.Compound 2,compound 3 and compound 5 were first obtained from the plant.Some compounds isolated from the effective fractions were screened,and 3 anti-tumor active compounds were obtained,they were compound 1,compound 3 and compound 4.Compound 1 could inhibit the proliferation and induce apoptosis of PC3 and B16 cells,which was a concentration-dependent manner and time-dependent manner.