ABSTRACT
In this study, nrDNA ITS sequences of Lycium cultivars were sequenced and used to test the existence of incomplete concerted evolution and pseudogenes. Together with 44 ITS sequences retrieved from GenBank, the pattern of base substitutions, GC content, 5.8S conserved motifs, the minimum free energy of secondary structures, nucleotide diversity and phylogenetic relationship of the samples were analyzed. While 83 of the 144 sequences were identified as pseudogenes, the results suggested a high degree of polymorphism and putative pseudogenes in Lycium, suggesting an incomplete concerted evolution of the ITS region. ITS polymorphism and pseudogene of Lycium were systematically tested for the first time. This research provides a references for ITS sequence to be used in the study of Lycium germplasm resources and DNA barcode identification.
ABSTRACT
Chuanxiong Qingfengteng mixture (CQM) is an analgesic developed based on clinical evidence and traditional Chinese medicine theory, which majorly consists of Ligusticum chuanxiong and Sinomenium acutum extracts. The current study aims to establish an UHPLC-UV method for the quantification of sinomenine and ligustrazine after CQM administration to rats, mice and cells, and to study the brain permeability of sinomenine and ligustrazine. The selectivity, linearity, accuracy, precision and stability of the established method demonstrated that it was suitable for the determination of sinomenine and ligustrazine in biological samples such as plasma, brain tissue and cellular fluid. After CQM was intravenously administered to rats and mice, both sinomenine and ligustrazine were detected in the brain from 5 min-2 h. The CSF/plasma partition coefficients (Kp, C/P) of each component were higher than those of brain tissue/plasma partition coefficient (Kp, B/P), the Kp, C/P and Kp, B/P of ligustrazine were higher than those of sinomenine. The concentrations between CSF and brain tissue were strongly correlated (Pearson's R>0.86, P<0.001). The unbound fraction in plasma of sinomenine and ligustrazine was 78.92% and 34.07%, respectively. The plasma protein binding rates displayed concentration-independent behavior within their respective in vivo concentration ranges. After CQM co-cultured with Caco-2 cell monolayers, the apparent permeability coefficient (Papp) of sinomenine and ligustrazine were 1.30×10-6 and 3.64×10-6 cm·s-1, respectively, following into the range of the intermediate and high permeability compounds. The efflux ratio (Papp(basolateral→apical)/Papp(apical→basolateral)) of sinomenine and ligustrazine were 0.67 and 0.85, respectively. When combined with P-glycoprotein inhibitor, the Papp of each component did not increase. In conclusion, the UHPLC-UV assay was successfully applied for the brain permeability study of CQM, the components of CQM can be quickly distributed to cerebrospinal fluid and pass through the blood-brain barrier. The brain permeability of ligustrazine is higher than that of sinomenine. The transmembrane transport of sinomenine and ligustrazine may not be affected by efflux transporters. All animal care and use complied with the Regulations for the Administration of Affairs Concerning Experimental Animals approved by the State Council of the People's Republic of China. All animal studies were implemented according to protocols, which were reviewed and approved by the Institutional Animal Care and Use Committee at Experimental Research Center, China Academy of Chinese Medical Sciences.