Using cell membrane chromatography and HPLC-TOF/MS method for in vivo study of active components from roots of Aconitum carmichaeli

An offline two-dimensional system combining a rat cardiac mascle cell membrane chromatography time-of-flight mass spectrometry （CMC-TOF/MS） with a high performance liquid chromatography time-of-flight mass spectrometry （HPLC-TOF/MS） was established for investigating the parent components and metabolites in rat urine samples after administration of the roots of Aconitum carmichaeli. On the basis of the analysis of the first dimension, retention components of the urine sample were collected into 30 fractions （one fraction per minute）. Then offline analysis of the second dimension was carried out. 34 compounds including 24 parent alkaloids and 10 potential metabolites were identified from the dosed rat urine, and then binding affinities of different compounds on cell membranes were compared and influences of some functional groups on activity were estimated with the semi-quantification and curve fitting method. As a result, binding affinities decreased along with the process of deacylation, debenzoylation and demethylation, which may be related to the alleviation of toxicity in the procedure of herb processing or metabolism. Moreover, some minor components in rat urine （Songorine, 14-benzoylneoline, Deoxyaconitine, etc. ） exerted relatively strong affinity on cell membranes are worth exploring. The results delivered by the system suggest that the CMC can be applied to in vivo study.

Screening of immune cell activators from Astragali Radix using a comprehensive two-dimensional NK-92MI cell membrane chromatography/C18 column/time-of-flight mass spectrometry system

Astragali Radix(AR)is a clinically used herbal medicine with multiple immunomodulatory activities that can strengthen the activity and cytotoxicity of natural killer(NK)cells.However,owing to the complexity of its composition,the specific active ingredients in AR that act on NK cells are not clear yet.Cell membrane chromatography(CMC)is mainly used to screen the active ingredients in a complex system of herbal medicines.In this study,a new comprehensive two-dimensional(2D)NK-92MI CMC/C18 column/time-of-flight mass spectrometry(TOFMS)system was established to screen for potential NK cell activators.To obtain a higher column efficiency,3-mercaptopropyltrimethoxysilane-modified silica was synthesized to prepare the NK-92MI CMC column.In total,nine components in AR were screened from this system,which could be washed out from the NK-92MI/CMC column after 10 min,and they showed good affinity for NK-92MI/CMC column.Two representative active compounds of AR,isoastragaloside Ⅰ and astragaloside IV,promoted the killing effect of NK cells on K562 cells in a dose-dependent manner.It can thus suggest that isoastragaloside Ⅰ and astragaloside Ⅳ are the main immunomodulatory components of AR.This comprehensive 2D NK-92MI CMC analytical system is a practical method for screening immune cell activators from other herbal medicines with immunomodulatory effects.

Development of 3-mercaptopropyltrimethoxysilane(MPTS)-modified bone marrow mononuclear cell membrane chromatography for screening anti-osteoporosis components from Scutellariae Radix

Osteoporosis is a bone metabolic disease caused by the imbalance between osteoblasts and osteoclasts due to excess osteoclastogenesis,manifesting in the decrease of bone density and bone strength.Scutellariae Radix shows good anti-osteoporosis activity,but the effective component is still unclear.Cell membrane chromatography(CMC)is a biological affinity chromatography with membrane immobilized on a silica carrier as the stationary phase.It can realize a dynamical simulation of interactions between drugs and receptors on cell membrane,which is suitable for screening active compounds from complex systems.In this study,the components of Scutellariae Radix with potential anti-osteoporosis activity through inhibiting the differentiation from bone marrow mononuclear cells(BMMCs)to osteoclast were screened by a BMMC/CMC analytical system.Firstly,a new3-mercaptopropyltrimethoxysilane(MPTS)-modified BMMC/CMC stationary phase was developed to realize covalent binding with cell membrane fractions.By investigating the retention time(tR)of the positive drug,the life span of the MPTS-modified CMC columns was significantly improved from 3 to 12 days.Secondly,6 components of Scutellariae Radix were screened to show affinity to membrane receptors on BMMCs by a two-dimensional BMMC/CMC-TOFMS analytical system.Among them,tectochrysin demonstrated the best anti-osteoporosis effect in vitro,which has never been reported.We found that tectochrysin could inhibit the differentiation of BMMCs into osteoclasts induced by receptor activator of nuclear factor-κB ligand(RANKL)and macrophage colony-stimulating factor(M-CSF)in a concentration-dependent manner in vitro.In vivo,it significantly reduced the loss of bone trabeculae in ovariectomized mice,and decreased the level of C-terminal cross-linking telopeptides of type 1 collagen(CTX-1),tartrate-resistant acid phosphatase 5 b(TRAP-5 b),interleukin 6(IL-6)in serum.In conclusion,tectochrysin serves as a potential candidate in the treatment of osteoporosis.The proposed twodimensional MPTS-modified BMMC/CMC-TOFMS analytical system shows the advantages of longlife span and fast recognition ability,which is very suitable for infrequent cell lines.

A high expression EGFR/cell membrane chromatography and online high performance liquid chromatography/mass spectrometry method for screening EGFR antagonists from Rhizoma Polygoni Cuspidati

The epidermal growth factor receptors(EGFRs)in some tumor cells are significant targets for drug discovery.In this work,we have developed an EGFR cell membrane chromatography and online high performance liquid chromatography/mass spectrometry system for screening active component from Rhizoma Polygoni Cuspidati.As a result,resveratrol from Rhizoma Polygoni Cuspidati was found to be the active component acting on EGFR like gefitinib.There was a good relationship between their inhibiting effects on EGFR secretion and HEK293 EGFR cell growth in vitro.The EGFR/CMC-online-HPLC/MS system demonstrated fast and effective characteristics for screening leading compounds from traditional Chinese medicine.

INHIBITORY EFFECTS OF THE ALKALOIDS FROM Radix Caulophylli ON THE PROLIFERATION OF HUMAN VASCULAR ENDOTHELIAL CELL

Objective To find an angiogenetic inhibitor from Radix Caulophylli (RC). Methods The extract of Radix Caulophylli was obtained by using 95% alcohol in water as solvent. Then, the total alkaloids of Radix Caulophylli was isolated from the extract by using a positive ion exchange resin column. An active part was found by a screening model of cell membrane chromatography (CMC) and further tested by the MTT method with emodin as a control. Results The total alkaloids of Radix Caulophylli was the active part by CMC and could significantly inhibit proliferation of ECV304 cells in MTT test. The inhibitory rate was 56.06% while the concentration of the total alkaloids of Radix Caulophylli was 19.63μg/mL. Conclusion The total alkaloids from Radix Caulophylli may be a new angiogenetic inhibitor, and mechanism of the total alkaloids on inhibitory angiogenesis still need to be further investigated.