| BACKGROUD&OBJECTTVE: Honokiol is one of the main chemical compositions of Chinese native medicine-Magnolia officinalis Rehd.et Wils. It has been reported that honokiol has the multi-way anti-tumor mechanism, for example, inducts cell multiplication and splits up, causes the cell to perish weakly, its mechanism is that it upwards presses to perish weakly factor Bid, declines damps perishes weakly factor Bcl-XL, as well as activates the p53 non-dependence and the plastochondria dependence Caspase circuit. Own to animal experiment, we need to find a better separation technology to purify honokiol. HSCCC is one kind of fluid-fluid disengaging gear, through many year improvements; it has been one kind of fast separation technology and one kind of separation natural medicine ideal equipment. SILAC technology: that is to add the isotope marked essential amino acids to the culture medium which lacks this amino acid, process cell metabolism with this culture medium culture cell, the mark amino acid fuses in the protein in, forms the numeral. It has provided and the good technology with the mass spectrum union for the quantitative proteomics. The purpose of this article is to apply HSCCC large-scale separation honokiol, and using the SILAC technology, honokiol treats the isotope mark liver cancer cell HepG2, through the mass spectrum appraisal, will appraise the protein regulation with the quantitative protein group's method which and the honokiol will cause, thus it will determine honokiol anti-tumor action mechanism.METHODS: Uses analysis HSCCC which English Brunei provided optimization separation conditions, including speed of flow, specimen handling density, specimen handling quantity. And the optimize condition will enlarge to preparation HSCCC in order to large-scale separate honokiol. Using honokiol treats liver cancer cell HepG2 which marked completely with leucine-d3, extracts protein, examines the total protein using the mass spectrum, takes the conservative proteinβ-actine as the standard, determins honokiol caused the regulative protein, which infers to honokil anti-tumor action mechanism.RESULTS: Using analysis HSCCC to optimize separation conditions, the chromatography conditions: The rotational speed is each minute 1800 revolutions; The speed of flow is each minute 2.5 milliliters; The specimen handling quantity is the separation column 5%; The specimen handling density is each milliliter 80 milligrams. These optimized conditions succeed utilization preparation HSCCC. In the large-scale separation purification honokiol and magonol, we use three kind of different density(20mg/ml, 75mg/ml, and 250 mg/ml) specimen handling, we separated 24.7g magonol which purity reached as high as 99.9% and 23.3g honokil which purity reached as high as 98.6%. We used 10μg/ml honokiol to treat HepG2 cell 24h, applying to mass spectrum appraised 290 proteins, in which the upward proteins accounted for 5.5%, the declined proteins accounted for 72.4%. There were 23 correlation proteins with cell perish. CONCLUSIONS: In this article, we succeeded have used the HSCCC large-scale purification honokiol. Apply to SILAC technology, we found the proteins which honokiol caused, in 23 correlation proteins with cell perish, we choosed Ras GTPase-activating-like protein IQGAP1(p195) and 94 kDa glucose-regulated protein(GRP94) carry on PCR and the Westen-blot confirmation. It confirmed that honokiol possibly through caused a series of proteins regulation to induce the cell to perish...
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