RESUMO
Objective A new hollow fiber liquid phase microextraction combined with high-performance liquid chromatography was developed to determine the concentration of berberine in rat plasma. Methods Parameters that affect the hollow fiber liquid phase microextraction processes were investigated and optimized. The optimized conditions were pH of donor and acceptor phases 12 and 2.0, respectively; extraction time 20 min; stirring speed 800 rpm; and addition of 10 % (w/v) salt. Under the optimized conditions, the preconcentration factor for berberine was 347. Moreover, a rapid and sensitive method was developed to determine berberine in rat plasma by HPLC. Results The calibration curve for berberine was linear in the range of 10-1000 ng/ml (r2≥0.9992). The limit of quantitation for the analyte was 10 ng/ml (S/N=10). The limit of detection for the analyte was 3.3 ng/ml (S/N=3). The intra-day and inter-day precision, and stability (RSD) were less than 6.3%. The average recovery was 96.7% ± 3.82%, and RSD was 4.82%. Conclusions The method is efficient, green, accurate and repeatable. It can be applied in determination of berberine in rat plasma.
RESUMO
To use Agrobacterium rhizogenes-induced hairy roots to create new germplasm of Dianthus caryophyllus, we transformed D. caryophyllus with A. rhizogenes by leaf disc for plant regeneration from hairy roots. The white hairy roots could be induced from the basal surface of leaf explants of D. caryophyllus 12 days after inoculation with A. rhizogenes ATCC15834. The percentage of the rooting leaf explants was about 90% 21 days after inoculation. The hairy roots could grow rapidly and autonomously in liquid or solid phytohormone-free MS medium. The transformation was confirmed by PCR amplification of rol gene of Ri plasmid and silica gel thin-layer chromatography of opines from D. caryophyllus hairy roots. Hairy roots could form light green callus after cultured on MS+6-BA 1.0-3.0 mg/L + NAA 0.1-0.2 mg/L for 15 days. The optimum medium for adventitious shoots formation was MS + 6-BA 2.0 mg/L + NAA 0.02 mg/L, where the rate of adventitious shoot induction was 100% after cultured for 6 weeks. The mean number of adventitious shoot per callus was 30-40. The adventitious shoots can form roots when cultured on phytohormone-free 1/2 MS or 1/2 MS +0.5 mg/L NAA for 10 days. When the rooted plantlets transplanted in the substrate mixed with perlite sand and peat (volume ratio of 1:2), the survival rate was above 95%.