ABSTRACT
The present study was designed to develop a practical strategy to tackle the problem of lacking standard compounds and limited references for identifying structure-related compounds in Streptocaulon griffithii Hook. f., especially those in trace concentrations, with a focus on antitumor activity. The cardiac glycosides (CGs)-enriched part was determined using in vitro bioactive assays in three cancer cell lines and then isolated using macroporous resins. The MS and MS/MS data were acquired using a high performance liquid chromatography coupled with hybrid quadrupole-time of flight (HPLC-Q-TOF-MS) system. To acquire data of trace compound in the extract, a multiple segment program was applied to modify the HPLC-Q-TOF-MS method. A mass defect filter (MDF) approach was employed to make a primary MS data filtration. Utilizing a MATLAB program, the redundant peaks obtained by imprecise MDF template calculated with limited references were excluded by fragment ion classification, which was based on the ion occurrence number in the MDF-filtered total ion chromatograms (TIC). Additionally, the complete cleavage pathways of CG aglycones were proposed to assist the structural identification of 29 common fragment ions (CFIs, ion occurrence number ≥ 5) and diagnostic fragment ions (DFIs, ion occurrence number < 5). As a result, 30 CGs were filtered out from the MDF results, among which 23 were identified. This newly developed strategy may provide a rapid and effective tool for identifying structure-related compounds in herbal medicines.
Subject(s)
Animals , Humans , Mice , A549 Cells , Apocynaceae , Chemistry , Cardiac Glycosides , Chemistry , Pharmacology , Toxicity , Cell Line, Tumor , Cell Survival , Chromatography, High Pressure Liquid , Computational Biology , Data Mining , Drugs, Chinese Herbal , Chemistry , Pharmacology , Inhibitory Concentration 50 , MCF-7 Cells , Molecular Structure , Plant Roots , Chemistry , Plants, Medicinal , Chemistry , Tandem Mass Spectrometry , WorkflowABSTRACT
The present study was designed to develop a practical strategy to tackle the problem of lacking standard compounds and limited references for identifying structure-related compounds in Streptocaulon griffithii Hook. f., especially those in trace concentrations, with a focus on antitumor activity. The cardiac glycosides (CGs)-enriched part was determined using in vitro bioactive assays in three cancer cell lines and then isolated using macroporous resins. The MS and MS/MS data were acquired using a high performance liquid chromatography coupled with hybrid quadrupole-time of flight (HPLC-Q-TOF-MS) system. To acquire data of trace compound in the extract, a multiple segment program was applied to modify the HPLC-Q-TOF-MS method. A mass defect filter (MDF) approach was employed to make a primary MS data filtration. Utilizing a MATLAB program, the redundant peaks obtained by imprecise MDF template calculated with limited references were excluded by fragment ion classification, which was based on the ion occurrence number in the MDF-filtered total ion chromatograms (TIC). Additionally, the complete cleavage pathways of CG aglycones were proposed to assist the structural identification of 29 common fragment ions (CFIs, ion occurrence number ≥ 5) and diagnostic fragment ions (DFIs, ion occurrence number < 5). As a result, 30 CGs were filtered out from the MDF results, among which 23 were identified. This newly developed strategy may provide a rapid and effective tool for identifying structure-related compounds in herbal medicines.
Subject(s)
Animals , Humans , Mice , A549 Cells , Apocynaceae , Chemistry , Cardiac Glycosides , Chemistry , Pharmacology , Toxicity , Cell Line, Tumor , Cell Survival , Chromatography, High Pressure Liquid , Computational Biology , Data Mining , Drugs, Chinese Herbal , Chemistry , Pharmacology , Inhibitory Concentration 50 , MCF-7 Cells , Molecular Structure , Plant Roots , Chemistry , Plants, Medicinal , Chemistry , Tandem Mass Spectrometry , WorkflowABSTRACT
<p><b>OBJECTIVE</b>To analyze the expression of midkine (MK) gene in acute leukemia patients, and explore the relationship between the gene and leukemia.</p><p><b>METHODS</b>The MK gene expression levels were detected by real-time quantitative RT-PCR (RQ-RT-PCR) in bone marrow (BM) of 181 acute leukemia (AL) patients and 31 normal controls.</p><p><b>RESULTS</b>MK gene was expressed in all AL patients, normal controls and AL patients in complete remission (CR). Compared with that in control group and CR group, MK gene expression was significantly increased in AL patients (P < 0.01 and P < 0.05, respectively). No statistical difference was found between CR group and control group. The expression of MK showed a notable increase in all B-ALL subtypes (including pro-B-ALL, common-B-ALL and pre-B-ALL) as well as in adult and childhood B-ALL patients (P < 0.01). Moreover, the gene expression in B-ALL was also significantly higher than that in TALL, HAL and FAB subtypes of AML (P < 0.01). In addition, M2 patients showed significantly increased in MK expression compared with that in normal controls (P < 0.01) and in other FAB subtypes of AML (P < 0.05). Median MK expression level in M3 patients was also significantly higher than that in normal controls (P < 0.05), but there was no statistical difference between M3 and other AL subtypes excepting for M2 and B-ALL. MK expression in CD34 positive patients was significantly higher than that in CD34 negative ones (P < 0.01) and within M2 patients, MK expression was higher in patients with t (8 ;21) than in those without the translocation (P < 0.05).</p><p><b>CONCLUSION</b>MK gene expression is increased with different levels in B-ALL, M2 and M3 patients, which provides novel insights into the leukemogenesis of acute leukemia.</p>