RESUMO
A simple and reliable high-performance liquid chromatographic (HPLC) method was developed for the simultaneous determination of five hydroxyanthraquinones (aloe-emodin, rhein, emodin, chrysophanol, and physcion) in Rhubarb and experimental animal bodies. A Zorbax SB-C18 column (250 mm x 4.6 mm i.d., 5 microm) and a methanol-0.5% acetic acid (85:15, v/v) mobile phase were used for the separation. The detection wavelength of a diode array detector (DAD) was set at 254 nm. Regression equations revealed a linear relationship (R2>0.9996) between the mass of hydroxyanthraquinones injected and the peak areas detected by DAD. The detection limits (S/N=3) ranged from 0.35 ng to 3.13 ng, and the recoveries ranged from 83% to 103% for different hydroxyanthraquinones. This method is simple, sensitive and suitable for the analysis of hydroxyanthraquinones in medicinal materials and pharmacological experiment samples.
RESUMO
Techniques, named two-step enrichment and double-time replica-plating method (TEDR), are described that allow a mutated population of Candida tropicalis to be enriched efficiently for mutants deficient in the alkane degradation pathway (Alk(-)) and to be selected easily for mutants increasing in the DCA (dicarboxylic acids) excretion pathway. After C. tropicalis was mutated with ethyl methane sulphonate and ultraviolet, the Alk(-) mutants were enriched (the first step enrichment, up to eightfold in one round of enrichment) by treatment with nystatin in medium SEL1-1. The mutagen-treated cells were then cultured in medium YPD containing chlorpromazine for further enriching (the second-step enrichment, up to threefold in one round) the mutants with an increasing capacity of alpha- and omega-oxidation. On the other hand, the Alk(-) mutants were readily isolated by the SEL1 replica-plating method by using alkane or glucose as the sole carbon source. A total of 43 Alk(-) mutants were isolated from 2x10(8) mutagen-treated cells. In the following steps, by using SEL2 replica plating, the screening studies showed that of the 43 Alk(-) mutants, 11 strains could accumulate DCA greatly from alkane, and strains 1-12 and 1-3, especially, could produce nearly three times as much DCA as the wild-type organism could. The results showed that the strains had more cytochrome P450 activity and a higher converting capacity of alkane.