Alkaloid Of Betacyanin Induces Apoptosis In Human Chronic Myeloid Leukemia Cell Line-K562

The extraction,purification,structure,standard,determination of betalains and inducesapoptosis in human chronic myeloid leukemia Cell line-K562activity in vitro were studied.The results were as follows:1、Optimization of ultrasonic extraction technology of Red Pigment from Fruits ofPhytolacca acinosa Roxb by orthogonal experimentsThe technique of ultrasonic extraction of Ph.americana L red pigment was studiedusing orthogonal test in this paper．The results showed that optimal ultrasonic extractionconditions were：extration30min, Solid-liquid ratio1:4(g:ml),pH value3and extrationtimes twice.Ultrasonic extraction method was simpler and showed higher efficiency butshorter extraction time, so suitable for large-scale production applications．2、Macroporous Adsorption Resin for the Extraction and Separation of Red Pigmen fromFruits of Phytolacca acinosa RoxbConsidering overall adsorption and desorption capacity, AB-8resin was selected outof5types of macroporous resins to purify the red pigment from fruits of Phytolaccaacinosa Roxb. It is the best conditions of isolation the red pigment from fruits ofPhytolacca acinosa Roxb. And the optimum process conditions for purifying the redpigment were determined as follows：pH as3, adsorption velocity2.0mL/min, solutionadsorbent as15%ethanol, elution velocity0.7mL/min； And the optimum processconditions for purifying the red pigment using ethanol precipitation were determined asfollows: eluate concentrated50time，concentrated solution：ethanol as1:10，precipitation3time. After AB-8macroporous resin purification, ethanol precipitation is not only able toremove most of the impurities, but also greatly enhance the pigment quality.3、Purification technology of Ph.americana L red pigment by Sephadex LH-20To optimize the purification technology of Ph.americana L red pigment by SephadexLH-20.And the optimum process conditions for purifying the red pigment were determinedas follows:sample volume as5.0mL, linear velocity as7.15cm/h, pH as3. Sephadex LH-20 is not only able to remove most of the impurities, but also greatly enhance the pigmentquality.4、Analysis and preparative technology of Ph.americana L red pigment by HPLCPurification technology of Ph.americana L red pigment by HPLC from SephadexLH-20.Conditions for preparative HPLC were as follows: Crest ODS PN：Co218050-212250（5μm,21.2mm×250mm）; linear gradient within60min18%MeOH in0.5%aqueous formic acid;10mL/min flow rate;2.0mL injection volume; detection at538nm; SPD-M10A detector (DAD), and operated at room temperature. Conditions foranalytical ware as follows: Sino Chrom ODS-Bp(5μm,4.6mm×150mm);Linear gradientelution was within60min from10to30%MeOH in0.5%aqueous formic acid, and within30min30%MeOH in0.5%aqueous formic acid;1.0mL/min flow rate;10μL injectionvolume; detection at538nm; SPD-M10A detector (DAD), and operated at roomtemperature.5、Identification of structures of betalains from Ph.americana L red pigmentOne compound was obtained by combined use of ultrasonic extraction,macroporousadsorptive resins, Sephadex LH-20and preparative liquid chromatography. It wasidentified as betanidin, by spectroscopic data (ultraviolet spectrum, electrosprayionization-mass spectrometry, nuclear magnetic resonance) analysis.6、Quality control of betalains.The optimum UV-vis and HPLC condition wase confirmed to determine the contentof betalains from Ph.americana L red pigment. The optimum UV-vis condition waseconfirmed to determine the content of betalains from Ph.americana L red pigment. A goodlinear relationship was established in the range0.01~0.05mg/mL for Ph.americana L redpigment with the regression equation y=1.9662x-5.17, r=0.9992; Conditions for analyticalwas as follows: Sino Chrom ODS-Bp(5μm,4.6mm×150mm);Linear gradient elution waswithin60min from10to30%MeOH in0.5%aqueous formic acid, and within30min30%MeOH in0.5%aqueous formic acid;1.0mL/min flow rate;10μL injection volume;detection at538nm; SPD-M10A detector (DAD), and operated at room temperature.Agood linear relationship was established in the range6.00～22.00μg for betanidin with theregression equation Y=1016425.225X+1824551.050，r=0.9996. It is simple and reliablefor the quantitative determination and quality control of betalains.7、betanin a betacyanin pigment purified from fruits of Ph.americana L inducesapoptosis in human chronic myeloid leukemia Cell line-K562In the present study the antiproliferative effects of betanin, a principle betacyanin pigment, isolated from the fruits of Ph.americana L, was evaluated on human chronicmyeloid leukemia cell line(K562). The results show dose and time dependent decrease inthe proliferation of K562cells treated with betanin with an IC50of6.395、5.304、4.796μMat24、48、72h. Further studies involving scanning and transmission electron microscopyrevealed the apoptotic characteristics such as chromatin condensation, cell shrinkage andmembrane blebbing. Agarose electrophoresis of genomic DNA of cells treated withbetanin showed fragmentation pattern typical for apoptotic cells. Flow cytometric analysisof cells treated with0、1、5、10μM betanin showed26.63%、29.05%、32.32%of cells insub G0/G1phase at24、48、72h. Cell surface antigen experiments show that betanin caninduce K562leukemia cell differentiation at IC505.30μM of48h.