| Objective: Isolating the anticancer active principle in the Caesalpinia sappan wood extracts, so as to observe the anticancer active principle's growth inhibiting effects and investigated the mechanism of apoptosis-inducing on bladder cancer cell line T24. Establishing the implantation nude mice and T739 mice models of bladder cancer, so as to observe the cancer inhibiting effect by the anticancer active principle's in bearing cancer animal.Methds: Crude extracts were extracted from Caesalpinia sappan wood with different solvents, Liquid chromatography was used to analyze the content of each essential component in the extractsion fractions; Trypan blue exclusion test was performed to detect the growth suppression rate of human bladder carcinoma cell line T24 treated by the extraction fractions at different time points (20, 40, 60, 80, 100min); The main component positively correlated with the cell suppression rate was separated out using repeated chromatography, thus the anticancer active monomer was obtained, with a purity above 92%; Its chemical constitution was determined using NMR (nuclear magnetic resonance), mass spectrum and infrared spectrum methods. Trypan blue exclusion test was performed to detect the inhibition of Brazilin on the growth of T24 cell lines in vitro cultured at different time points (4,8,24,48h). After exposure to different concentrations of Brazilin, homogeneous bioluminescence assay was used to detect the inhibitory action of Brazilin on T24 cells and the caspase-3,9 activity. Cellular apoptosis was measured by flow cytometry (FCM) and observed by laser scanning confocal microscope. T24 and BTT cell lines were inoculated subcutaneously and intraperitoneally into nude mice and T739 mice, and both models were randomly divided into control group, the the extract of Caesalpinia sappan L(the content of Brazilin is above 50%) group and mitomycin C (MMC) group; Record the tumor formation time in mice, and measure the length and width so as to calculate the volume of tumor and the tumor inhibition ratio in the models of subcutaneously implantation; Recorded survival time of intraperitoneal implantation mice so as to calculate the life extension ratio. compared the mean survival time of control group with the the extract of Caesalpinia sappan L group and MMC group.Result: Study on anticancer active principle in lignum Caesalpiniae sappan: Among the extraction fractions, R12 has a positive correlation with the cell suppression rate(r100min=0.941, P<0.001); Brazilin is the key component in R12. Cell experiment: Trypan blue exclusion test results showed the different effects of cell growth after treatment with Brazilin, cells were not affected at 4 hour time point when Brazilin was at or below 25μg/ml (P>0.05), after 8 hours, Brazilin could significantly inhibit the proliferation of T24 cells, the inhibitory rates of the Brazilin at 25,50,100,200μg/ml against T24 cells were 43.19%,60.73%,86.38% and 93.89% (P<0.05), after exposure to 50μg/ml of Brazilin, the inhibition ration of T24 cells for increasing times (4,8,24,48h), staining increased (52.72%,60.73%,91.77%,96.41%) (P<0.05). The activity of caspase-3,9 increased slightly when Brazilin was at 25μg/ml, compared with the control group, there was no statistical significance in the differences (P>0.05). Cells were treated with Brazilin for 16 hours, with an increase of the concentration of Brazilin within the range of (7.5-60)μg/ml, the apoptosis ratio in turn showed a upward trend of 0.15%,1.35%,2.91%,34.76%. It could be seen by laser scanning confocal microscope that the apoptosis occurred in the cells.Animal experiment results showed that when the extract of Caesalpinia sappan L (the content of Brazilin is above 50%) was given at the dosage of 400, 300 and 200mg/Kg, the life extension ratio was 58.42%, 56.90% and 51.50% in the T24-bearing nude mice, respectively (P<0.05),the life extension ratio was 11.80%, 4.32% and 10.77% in the BTT-T739 mice (P>0.05), respectively. As observation was terminated in the models of Subcutaneous implantation in nude mice, the mean volume of control groups was 1.092±0.403cm~3, the extract of Caesalpinia sappan L groups (300mg/Kg) and MMC groups were respectively 0.656±0.117cm~3, 0.238±0.853cm~3. Comparisons of the mean volume of control groups with the volume of the extract of Caesalpinia sappan L groups and MMC groups have statistical significant difference (P<0.05).the inhibitory rates of the the extract of Caesalpinia sappan L groups and MMC groups against the subcutaneous tumor were respectively 40.25% and 69.78%.Conclusion: Brazilin is one of the essential anticancer principles in Caesalpinia sappan wood. Brazilin can effectively inhibit the proliferation of T24 cells and induce apoptosis in a dose dependent manner. the survival time of nude mice was extended and the growth of the Subcutaneous tumor in T739 mice were markedly inhibited by the extract of Caesalpinia sappan L (the content of Brazilin is above 50%) in a certain dose, but the drug have no obvious effects to extend the survival time of BTT-T739 mice.
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