Study On The Antitumor Activity And Its Functional Mechanism Of Chemical Compounds From Caesalpinia Sappan L.

Objective:The chemical compounds in Caesalpinia sappan L.,and their activities of antitumor and inhibtion on topoisomeraseâ… in vitro were investigated.It was supposed that the effective antitumor chemical entity and its functional mechanism would be elucidated in this thesis.Methods:(1)The 95%ethanol extract of Caesalpinia sappan L.was partitioned sequentially using ethyl acetate and butanol solvents.The butanol fraction was subjected repeatedly to Si-gel column chromatography and yielded fourteen chemical constituents,and the isolated compounds were structural elucidated by spectroscopy methods,such as nuclear magnetic resonance,infrared and mass spectroscopy.(2)Antitumor experiment:HL-60(human promyelocytic leukemia)and SWO-38 (human brain neuroglioma)cell lines with the tested samples at various concentrations was kept in an incubator for 24 h at 37℃in a humidified atmosphere of 95%air and 5%CO₂, respectively.After staining by addition of CCK-8 to each well,the plates were further incubated for 2h.The absorbance was read out on a microplate reader using a test wavelength of 450 nm.The dose response curve was plotted for each tested sample,and the medium cytotoxicity concentration(TC₅₀)was calculated. Brazilein,sappanchalcone and 3-deoxysappanchalcone were tested in experiments.(3)Reactions(20μL)containing 0.5μg of supercoiled pBR322 DNA,1 unit of topoisomeraseâ… with or without 0.5μL of tested samples at various concentrations were incubated at 37℃.After 30 min,reactions were terminated by the addition of 4μL of solution containing 5%sodium dodecyl sulfate(SDS)and 2.5 mg of proteinase K per ml. Following an additional incubation at 37℃for 30 min,the samples were electrophoresed through a 1.0%agarose gel in TBE buffer.The gel was stained with ethidium bromide, destained in water,and photographed on a UV transilluminator followed by densitometric analysis.Enzyme activity was measured in terms of the percentage of substrate DNA converted to product.Brazilein,sappanchalcone and protosappanin A were tested in experiments.Results:(1)Fourteen chemical compounds were isolated from Caesalpinia sappan L.,and their structures were identified as 3'-deoxy-4-O-methylepisappanol,(+)-lyoniresinol,(+)-(8S, 8'S)-bisdihydrosiringenin,protosappanin A,protosappanin B,isoprotosappanin B,sappan chalcone,3-deoxysappanchalcone,sappanone B,3-deoxysappanone B,brazilin,3'-O-methylbrazilin,brazilein and palmitic acid。(2)Sappanchalcone,brazitein and 3-deoxysappanchalcone could inhibit the growth of HL-60 and SWO-38 cells.The TC₅₀of brazilein and 3-deoxysappanchalcone on HL-60 cells was 19.05 and 5.44μM,respectively.The TC₅₀of sappanchalcone,brazilein and 3-deoxysappanchalcone on SWO-38 cells was 398.77,128.80,340.44μM,respectively. Compared with 189.57μM,the TC₅₀value for positive control drug adriamycin,128.80μM showed brazilein a potential antitumor agent.(3)Brazilein showed the activity of topoisomeraseâ… inhibitor,and its minimum inhibitory concentration(MIC)was less than 100μg/mL in vitro.However, sappanchalcone and protosappanin A didn't showed obvious inhibition on topoâ… .Conclusion: (1)The derivatives of five main structural types,such as brazilin,chalcone, protosappanin,homisoflavonoid and phenylpropanoid in C.sappan were obtained successfully.(2)3'-Deoxy-4-O-methylepisappanol was a novel compound,and(+)-(8S,8'S)-bisdi hydrosiringenin was isolated from family Caesalpiniaceae for the first time.(3)As far as the activiey of antitumor and topoâ… inhibition was concerned,brazilein was the active compound.The TC₅₀of brazilein on HL-60 and SWO-38 cell lines was 19.05 and 128.80μM,and its MIC on topoâ… was less than 100μg/mL.The ruselts indicated that brazilein may be the main antitumor compounds of C. sappan,due to its high content in plant tissue and its planarized molecular structure,the latter make it an easy interaction between DNA strands and act as a topo inhibitor. Therefore,brazilein,as a topo inhibitor,inhibites tumor cells proliferation is one of antitumor functional mechanisms of C.sappan L.