Chemical profiling of Fritillariae thunbergii Miq prepared by different processing methods reveals two new quality markers: Zhebeininoside and imperialine-3-ß-D-glucoside.

ETHNOPHARMACOLOGICAL RELEVANCE: Fritillariae thunbergii Miq (FTM)exhibit versatile biological activities including the significant antitussive and expectorant activities. As a herbal medicine, the therapeutic effects of FTM may be expressed by multi-components which have complicated integration effects on multi-targets. With the time going, the different processing methods of FTM has been changed a lot. Thus，the study described the effect of processing methods to FTM and its quality. MATERIAL AND METHOD: Studies were undertaken by using UHPLC-LTQ Orbitrap MS and pharmacodynamic models. All reagents were involved of analytical grade. While a HPLC-ELSD's method has been developed and validated, a certified Quality System is conformed to ICH requirements. The experimental animals followed the animal welfare guidelines. AIM OF THE STUDY: We aimed to found the differences after the different processing methods of FTM, and to demonstrate the changes could be selected as quality control indicators, and established a method for simultaneous determination of these for quality control. RESULTS: we have previously found two new steroidal alkaloids: zhebeininoside and imperialine-3-ß-D-glucoside from the different processing methods of FTM, which is the difference between the different processing methods of FTM, mainly on the steroidal alkaloids. The activity analysis of zhebeininoside, imperialine-3-ß-D-glucoside, verticine and verticinone showed that the mouse model of cough expectorant has antitussive effect. The positive drug selected was dextromethorphan syrup. The positive group showed biological activity, but the blank group showed nothing. The model group showed illness which means that the model was effective. There are two ways of the mechanism of action of the expectorant action which can make sputum thin, reduce its viscosity, and be easy to cough up, or can accelerate the movement of mucous cilia in the respiratory tract and promote the discharge of sputum. In our study, the content of phenol red was significantly reduced in the administration group. CONCLUSIONS: To sum up, our results suggest that zhebeininoside and other three components cloud be selected as quality control indicators, and a method for simultaneous determination of zhebeininoside and other three components was established for quality control.

Steroidal alkaloids isolated from Veratrum grandiflorum Loes. as novel Smoothened inhibitors with anti-proliferation effects on DAOY medulloblastoma cells.

Constitutive activation of Hedgehog (Hh) pathway is intimately related with the occurrence and development of several malignancies, such as medulloblastoma (MB) and other tumors. Therefore, small molecular inhibitors of Hh pathway are urgently needed. In this study, three new steroidal alkaloids, ⊿5 (20R, 24R) 23-oxo-24-methylsolacongetidine, ⊿5 (20S, 24R) 23-oxo-24-methylsolacongetidine and veralinine 3-O-α-l-rhamnopyranosyl-(1 â†’ 2)-ß-D-glucopyranoside, together with six known alkaloids, 20-epi-verazine, verazine, protoverine 15-(l)-2'-methylbutyrate, jervine, veramarine and ß1-chaconine, were isolated and determined from Veratrum grandiflorum Loes. The dual-luciferase bioassay indicated that all compounds exhibited significant inhibitions of Hh pathway with IC50 values of 0.72-14.31 µM against Shh-LIGHT 2 cells. To determine whether these Hh pathway inhibitors act with the Smoothened (Smo) protein, which is an important oncoprotein and target for this pathway, BODIPY-cyclopamine (BC) competitive binding assay was preferentially performed. Compared with BC alone, all compounds obviously reduced the fluorescence intensities of BC binding with Smo in Smo-overexpression HEK293T cells through fluorescence microscope and flow cytometer. By directly interacting with Smo, it revealed that they were actually novel natural Smo inhibitors. Then, their anti-tumor effects were investigated against the human MB cell line DAOY, which is a typical pediatric brain tumor cells line with highly expressed Hh pathway. Interestingly, most of compounds had slight proliferation inhibitions on DAOY cells after treatment for 24 h same as vismodegib, while ß1-chaconine showed the strongest inhibitory effect on the growth of DAOY with IC50 value of 5.35 µM. In conclusion, our studies valuably provide several novel natural Smo inhibitors for potential targeting treatment of Hh-dependent tumors.

[Diterpenoids as PPARγ agonists from Siegesbeckia pubescens and their anti-inflammatory effects in vitro].

This study aims to investigate the PPARγ agonists isolated from the aqueous extract of Siegesbeckia pubescens( SPA) and their anti-inflammatory activities in vitro. The 293 T cells transfected transiently with PPARγ recombinant plasmid were used as a screening model to guide the isolation of PPARγ activitating components,and then PPARγ activities were measured by double luciferase reporter gene assay. The chemical structures were identified by chromatography or spectroscopic techniques. Furthermore,a UC inflammatory model in vitro was established on HT-29 cells by stimulating with TNF-α． The mRNA levels and secretion of proinflammatory cytokines on HT-29 cells,such as IL-1ß,TNF-α,IL-8,were detected by RT-PCR and ELISA. The results showed that five diterpenoids were obtained from the fraction D_(50) with the strongest PPARγ activity among others in SPA,and determined as kirenol( 1),darutigenol( 2),enantiomeric-2-ketone-15,16,19-three hydroxypinomane-8( 14)-ene-19-O-ß-D-glucoside( 3),darutoside( 4),enantiomeric-2-ß,15,16,19-four hydroxypinomane-8( 14)-ene-19-O-ß-D-glucoside( 5),respectively. All the compounds exhibited active effects on PPARγ in a concentration-dependent manner( P<0. 01). In addition,compound 1 significantly inhibited the expression of IL-1ß mRNA and secretion of IL-8 on HT-29 cells inflammation model( P<0. 001); both compounds 2 and 3 effectively inhibited the expression of IL-1ß,TNF-α,IL-8 mRNA and secretion of IL-8( P<0. 01 or P<0. 001),although at different extent; compound 4 significantly inhibited the expression of IL-1ß and TNF-α mRNA( P<0. 01 or P<0. 001),while compound 5 inhibited the expression of IL-1ß mRNA obviously( P<0. 001). In conclusion,the diterpenoids 1-5 isolated from S. pubescens have the PPARγ activation activities and potential effects of anti-UC in vitro.

A novel allosteric site in casein kinase 2α discovered using combining bioinformatics and biochemistry methods.

Casein kinase 2 (CK2) is a highly pleiotropic serine-threonine kinase, which catalyzed phosphorylation of more than 300 proteins that are implicated in regulation of many cellular functions, such as signal transduction, transcriptional control, apoptosis and the cell cycle. On the other hand, CK2 is abnormally elevated in a variety of tumors, and is considered as a promising therapeutic target. The currently available ATP-competitive CK2 inhibitors, however, lack selectivity, which has impeded their development in cancer therapy. Because allosteric inhibitors can avoid the shortcomings of conventional kinase inhibitors, this study was aimed to discover a new allosteric site in CK2α and to investigate the effects of mutations in this site on the activity of CK2α. Using Allosite based on protein dynamics and structural alignment, we predicted a new allosteric site that was partly located in the αC helix of CK2α. Five residues exposed on the surface of this site were mutated to validate the prediction. Kinetic analyses were performed using a luminescent ADP detection assay by varying the concentrations of a peptide substrate, and the results showed that the mutations I78C and I78W decreased CK2α activity, whereas V31R, K75E, I82C and P109C increased CK2α activity. Potential allosteric pathways were identified using the Monte Carlo path generation approach, and the results of these predicted allosteric pathways were consistent with the mutation analysis. Multiple sequence alignments of CK2α with the other kinases in the family were conducted using the ClustalX method, which revealed the diversity of the residues in the site. In conclusion, we identified a new allosteric site in CK2α that can be altered to modulate the activity of the kinase. Because of the high diversity of the residues in the site, the site can be targeted using rational drug design of specific CK2α inhibitors for biological relevance.

How calcium inhibits the magnesium-dependent kinase gsk3ß: a molecular simulation study.

Glycogen synthase kinase 3ß (GSK3ß) is a ubiquitous serine/threonine kinase that plays a pivotal role in many biological processes. GSK3ß catalyzes the transfer of γ-phosphate of ATP to the unique substrate Ser/Thr residues with the assistance of two natural activating cofactors Mg(2+). Interestingly, the biological observation reveals that a non-native Ca(2+) ion can inhibit the GSK3ß catalytic activity. Here, the inhibitory mechanism of GSK3ß by the displacement of native Mg(2+) at site 1 by Ca(2+) was investigated by means of 80 ns comparative molecular dynamics (MD) simulations of the GSK3ß···Mg(2+)-2/ATP/Mg(2+) -1 and GSK3ß···Mg(2+)-2/ATP/Ca(2+)-1 systems. MD simulation results revealed that using the AMBER point charge model force field for Mg(2+) was more appropriate in the reproduction of the active site architectural characteristics of GSK3ß than using the magnesium-cationic dummy atom model force field. Compared with the native Mg(2+) bound system, the misalignment of the critical triphosphate moiety of ATP, the erroneous coordination environments around the Mg(2+) ion at site 2, and the rupture of the key hydrogen bond between the invariant Lys85 and the ATP O(ß2) atom in the Ca(2+) substituted system were observed in the MD simulation due to the Ca(2+) ion in active site in order to achieve its preferred sevenfold coordination geometry, which adequately abolish the enzymatic activity. The obtained results are valuable in understanding the possible mechanism by why Ca(2+) inhibits the GSK3ß activity and also provide insights into the mechanism of Ca(2+) inhibition in other structurally related protein kinases.