RESUMO
Medicinal plants with complex matrices are endowed with a wide scope of biological activities. The separation, quantification, characterization and purification of bioactive components from herbal medicine extracts have always challenged analysts. Fortunately, the advancement of various emerging techniques has provided potent support for improving the method selectivity, sensitivity and run speeds in medicinal plant analyses. In recent years, the advent of new-generation supercritical fluid chromatography (SFC) instruments and a wide diversity of column chemistries, coupled with the intrinsic technical features of SFC, have made it an alternative and prominent analytical platform in the medicinal plant research area. This work aims to give a comprehensive overview of the fundamentals, technical advancement and investigating parameters of SFC in combination with three prevalent detectors. Moreover, the latest research progress of SFC applications in medicinal plant analyses is illuminated, with focus on herbal medicine-related SFC papers on the analytical and preparative scale that were published during the period of 2012 to December 2018. The most relevant applications were classified based on the constituents to be analysed. As for the respective research cases, analytical protocols and data processing strategies were provided, along with the indicated restrictions or superiority of the method; thus, the current status of SFC in medicinal plant analysis was presented.
Assuntos
Cromatografia com Fluido Supercrítico/métodos , Compostos Fitoquímicos/análise , Plantas Medicinais/química , Cromatografia com Fluido Supercrítico/instrumentaçãoRESUMO
AIM: To express human soluble complement receptor type 1(sCR1)protein using ferment cell secreting type carrier and study the extraorgan biologic activity of recombinant human sCR1 fusion protein. METHODS: Total human RNA was extracted from peripheral blood. The full length cDNA of human sCR1 gene was obtained by RT-PCR and them, cloned into Pichia pastoris eukaryotic expression vector pPIC9k to construct the recombinant plasmid pPIC9k-sCR1 containing human sCR1.After identified by DNA sequencing, the recombinant plasmid pPIC9k-sCR1 was transformed into Pichia pastoris SMD1168. The ferment cell line of the recombinant sCR1 which was chosen by G418 resistance was identified by PCR, After methanol induction, the expressed protein products were verified by SDS-PAGE and Western blot, purified by Ni(2+)-NTA agarose affinity chromatography, and its biologic activity was identified. RESULTS: The obtained Pichia pastoris secretion type yeast carrier pPIC9k-sCR1 was chosen by G418 and identified by PCR to get a highly copied and integral recombinant ferment cell line. The recombinant human sCR1 fusion protein was expressed by yeast cells containing pPIC9k-sCR1 induced by methanol. It was a protein band about M(r) 31 000 in gel, which could be identified by CD35 of anti-sCR1 protein monoclonal antibody with Western blotting technique. The highly purified sCR1 fusion protein and its biologic activity were detected obtained by Ni(2+)-NTA agarose affinity chromatography. CONCLUSION: The recombinant human sCR1 fusion protein can be highly expressed in the Pichia pastoris expression system, which resembles the human natural protein's antigenicity and biologic activity.