Rapid flavonoid-focused sub-chemome characterization of Draconis Sanguis using UPLC-Q-TOF-MS in combination with molecular weight imprinting and mass defect filtering / 中国中药杂志

Draconis Sanguis is a precious Chinese medicinal material for activating blood and resolving stasis, and its effective components are flavonoids. However, the structural diversity of flavonoids in Draconis Sanguis brings great challenges to the in-depth chara-cterization of its chemical composition profiles. To clarify the substance basis of Draconis Sanguis, ultra-high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry(UPLC-Q-TOF-MS) was used in this study to acquire MS data of Draconis Sanguis. The molecular weight imprinting(MWI) and mass defect filtering(MDF) were developed for rapid screening of flavonoids in Draconis Sanguis. Full-scan MS and MS~2 were recorded within the mass range m/z 100-1 000 in positive ion mode. Accor-ding to previous literature, MWI was employed to hunt for reported flavonoids in Draconis Sanguis, and the mass tolerance range of [M+H]~+ was set as ±10×10~(-3). A five-point MDF screening frame was further constructed to narrow the screening range of flavonoids from Draconis Sanguis. Combined with diagnostic fragment ions(DFI) and neutral loss(NL) as well as mass fragmentation pathways, 70 compounds were preliminarily identified from the extract of Draconis Sanguis, including 5 flavan oxidized congeners, 12 flavans, 1 dihydrochalcones, 49 flavonoids dimers, 1 flavonoids trimer and 2 flavonoid derivatives. This study clarified the chemical composition of flavonoids in Draconis Sanguis. Moreover, it also showed that high-resolution MS combined with data post-processing methods such as MWI and MDF could achieve rapid characterization of the chemical composition in Chinese medicinal materials.

Isomeric discrimination of kaempferol versus luteolin by online energy-resolved mass spectrometry / 中国中药杂志

Isomers are widely distributed in Chinese herbal medicines,and can be discriminated by energy-resolved mass spectrometry( ER-MS). However,ER-MS was performed through direct injection of reference compounds with syringe pump,which encountered a significant technical barrier for high-throughput and automated measurements. Herein,online ER-MS was conducted using LC-MS platform,and a pair of isomers,kaempferol vs luteolin,were employed as a case study to illustrate and assess the utility of online ER-MS for isomeric discrimination. High-resolution tandem mass spectrometry data of both flavonoids were acquired on LC-QE-Orbitrap-MS,and the fragmentation pathways responsible for the primary fragment ions were proposed. The primary signal in MS1 occurred at m/z 285( [M-H]-),and the primary signals of either compound generated by retro-Diels-Alder fragmentation were observed at m/z 151 and 133. The spectral information was subsequently transferred onto LC-Qtrap-MS platform to carry out online ER-MS. Two precursor-to-product ion transition candidates were constructed as m/z 285>151 and 285>133,and either afterward derived a set of pseudo-ion transitions( PITs) and so forth,exactly corresponding to a series of progressive collision energies( eg-5,-8,-11 e V,and so on). All PITs were typed into the monitoring list of multiple reaction monitoring program to generate the peak area datasets. Either dataset was normalized using the highest values in the set and imported into Graph Pad Prism software to plot the Gaus-sian-shaped curve that was termed as the break-down graph. The apex of the regressive curve was termed as optimal collision energy( OCE). The OCE values corresponding to m/z 285>151 were calculated as-29. 06 e V and-35. 71 e V for kaempferol and luteolin,respectively. In the case of m/z 285>133,the OCEs were yielded as-44. 15 e V for kaempferol and-49. 01 e V for luteolin. With re-ference to their chemical structures,the location of hydroxyl group was regarded to be responsible for the differences of either m/z 285>151 or 285>133 between the isomers,attributing to their different bond properties. Above all,online ER-MS offers an eligible tool for isomeric discrimination,and provides meaningful information for the accurate chemical composition characterization based on LC-MS,which is not limited to Chinese herbal medicines.

Molecular mechanism of BMSC intracerebral transplantation in impro-ving learning and memory abilities of AD mice / 中国病理生理杂志

AIM:To investigate the effect of bone marrow mesenchymal stem cell(BMSC) transplantation on learning and memory abilities and pathological changes of Alzheimer disease (AD) mice and the molecular mechanisms. METHODS:C57/BL6 wild-type (WT) and transgenic(Tg) mice were randomly divided into 4 groups:WT/PBS group, WT/BMSCs group,Tg/PBS group and Tg/BMSCs group. The mice were administered with PBS or BMSCs via intracere-broventricular injection. Spatial learning and memory abilities of the mice were evaluated by Morris water maze test on the 3rd day after surgery. Real-time PCR was applied to detect the mRNA expression of CX3C chemokine ligand 1 (CX3CL1),CX3C chemokine receptor 1 (CX3CR1), IL-1β, TNF-α, Nurr1, YM1, insulin-degrading enzyme (IDE) and matrix metalloproteinase 9(MMP9). The protein levels of CX3CL1 and Aβ42 were measured by ELISA. Western blot was used to detect the protein expression of postsynaptic density protein 95 (PSD95) and synaptophysin (SYP). RE-SULTS:The transplanted BMSCs were observed near the hippocampus of APP/PS1 mice on the 10th postoperative day. The escape latency of the mice in Tg/PBS group was significantly longer than that in the WT/PBS mice(P<0.05). Com-pared with Tg/PBS group,the escape latency of Tg/BMSCs group was significantly shorter (P<0.05), and the mRNA and protein levels of CX3CL1 in Tg/BMSCs group were significantly higher than those in Tg/PBS group (P<0.01). The results of immunohistofluorescence staining showed that BMSC transplantation promoted the activation of microglia in the brain of WT and Tg mice. The mRNA expression of YM1 was up-regulated in WT/BMSCs group and Tg/BMSCs group (P<0.05). Compared with WT/PBS mice, the mRNA expression of TNF-α in the cortex and hippocampus of Tg/PBS group was significantly increased (P<0.05),and the mRNA expression of Nurr1 in the cortex was significantly decreased (P<0.01). Meanwhile,the mRNA expression of TNF-α in the cortex of Tg/BMSCs mice was decreased(P<0.01) and the mRNA expression of CX3CR1 and Nurr1 was up-regulated compared with Tg/PBS group (P<0.05). The results of Western blot showed that the protein levels of PSD95,p85,p110 and p-Akt in Tg/BMSCs group were significantly higher than those in Tg/PBS group (P<0.05). Finally, BMSC transplantation reduced the protein level of Aβ42 in APP/PS1 mice(P<0.05), and increased the mRNA expression of IDE and MMP9 in the hippocampus (P <0.05). CONCLU-SION:BMSC transplantation modulates neuroinflammatory responses and promotes neuroprotective factor and synaptic pro-tein expression,thus improving the learning and memory abilities in the APP/PS1 mice,which may be achieved by up-reg-ulating the expression of CX3CL1.