| Objective:The experimental research shows that the Flavonoids from Spina Gleditsiae(FSG) has a wide variety of tumor cell proliferation has obvious inhibition. studies on purification methods of ethanol extracts of the FSG such as through different solvents reflux extraction and silica gel column chromatography gradient elution, then determine the effect of restrain four kinds of tumor cells in vitro proliferation by CCK-8determination method, screening the constituent which been a higher inhibition rate and after further purification this one again, for the late subject to further purification and preparation of scientific research to provide effective monomer experiment basis of FSG.Methods:In different polarity of the organic solvent extract FSG total of traditional Chinese medicine backflow of different ingredients, get five different components of the extracted parts samples, in vitro culture HepG2human liver cell lines, Bel-7402liver cancer cell lines, K562people leukemia cell lines, HCT116colon cancer cell lines containing10%people in the womb of bovine serum RPMI1640medium, At37℃, saturated humidity5%CO2training in the box.Take logarithm the growing period of cells, adjust the right cellular levitation liquid density, vaccinations96hole in respectively training board, with different concentration (12.5,25,50,100,200)μ g·mL-1of different drug components and purification processing after24hours and48hours respectively, through CCK-8method to determine the effect of different parts of FSG separate product in vitro four tumor cell proliferation, chose suppress tumor proliferation were the higher rate of composition, to component silica gel column further purification again, with organic solvent gradient elutiononce again, to obtain the different purification samples, through CCK-8method to determine the effect of different parts of FSG separate product in vitro four tumor cell proliferation once again, further to screening suppress the higher rate of composition of the tumor cell proliferation. With statistical methods processing and analyzing data.Results:In the process of reflux extraction,polarity from small to large r in accordance with the order of sequence respectively as Petroleum ether, ethyl acetate,1-butanol,95%ethanol, pure water, five kinds of solvent to FSG reflux extraction to get five product, among those components, the tumor cells inhibition rate has good concentration-response relationship could be found by the n-butanol and95%ethanol extract in (12.5,25,50,100,200) μg·mL-1to HepG2concentrations, Bel-7402, K562, HCT116, especially in (100,200)μg·mL-1got very significant. The highest inhibition rate of N-butanol and95%ethanol in four of the cancer cells are (62.00±6.12)、(65.00±7.45);(43.00±9.34)、(76±13.22)、(94.00±3.74)、(93.00±5.38)、(92.00±6.32)、(95.00±12.11)。Select n-butanol and95%ethanol to the backflow product silica gel column as purification of the sample, five different elution compositions would be got, through CCK-8method to determine the effect of different parts of FSG separate product in vitro four tumor cell proliferation, chose suppress tumor proliferation were the higher rate of composition, to further purification.The components were of the tumor cell proliferation inhibitory effect with the increase of concentration increases, present a good concentration-response relationship, P<0.05, with statistical significance.Conclusion:The heating reflux extraction-silica gel column chromatography of purification process route to FSG separation and purification, some ingredients of purified antitumor activity obvious, and has a certain amount of Concentration-response relationship, which indicate that a single component by some or a single component of Spina Gleditsiae is relatively obvious antitumor activity and these single component can be out of extraction and separation, the antitumor activity ingredients of FSG further purification, screening and Spina Gleditsiaeantitumor activity in the extraction of research have preparation monomer far-reaching significance. |
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