ABSTRACT
Gambogic acid (GA) is an anticancer agent in phase ‖b clinical trial in China but its mechanism of action has not been fully clarified. The present study was designed to search the possible target-related proteins of GA in cancer cells using proteomic method and establish possible network using bioinformatic analysis. Cytotoxicity and anti-migration effects of GA in MDA-MB-231 cells were checked using MTT assay, flow cytometry, wound migration assay, and chamber migration assay. Possible target-related proteins of GA at early (3 h) and late stage (24 h) of treatment were searched using a proteomic technology, two-dimensional electrophoresis (2-DE). The possible network of GA was established using bioinformatic analysis. The intracellular expression levels of vimentin, keratin 18, and calumenin were determined using Western blotting. GA inhibited cell proliferation and induced cell cycle arrest at G2/M phase and apoptosis in MDA-MB-231 cells. Additionally, GA exhibited anti-migration effects at non-toxic doses. In 2-DE analysis, totally 23 possible GA targeted proteins were found, including those with functions in cytoskeleton and transport, regulation of redox state, metabolism, ubiquitin-proteasome system, transcription and translation, protein transport and modification, and cytokine. Network analysis of these proteins suggested that cytoskeleton-related proteins might play important roles in the effects of GA. Results of Western blotting confirmed the cleavage of vimentin, increase in keratin 18, and decrease in calumenin levels in GA-treated cells. In summary, GA is a multi-target compound and its anti-cancer effects may be based on several target-related proteins such as cytoskeleton-related proteins.
Subject(s)
Humans , Antineoplastic Agents , Pharmacokinetics , Apoptosis , Breast Neoplasms , Drug Therapy , Metabolism , Calcium-Binding Proteins , Genetics , Cell Line, Tumor , Cell Migration Assays , Cell Migration Inhibition , Cell Proliferation , Computational Biology , Methods , Cytoskeleton , Metabolism , Electrophoresis, Gel, Two-Dimensional , Flow Cytometry , Gene Expression , Keratin-18 , Genetics , Oxidation-Reduction , Protein Biosynthesis , Protein Transport , Proteomics , Methods , Transcription, Genetic , Ubiquitin-Specific Proteases , Pharmacokinetics , Vimentin , Genetics , Xanthones , PharmacokineticsABSTRACT
To determine 13 flavonoids and glycyrrhizic acid in licorice (Glycyrrhiza spp.), several samples from different areas were examined by HPLC-DAD analysis. The analysis was performed on a Zorbax Extend-C18 (250 mm × 4.6 mm, 5 μm) column connected with a Zorbax Extend guard column (20 mm × 4.6 mm, 5 μm). The mobile phase consisted of (A) acetonitrile and (B) 0.026% aqueous H3PO4 (VV) using a gradient elution of 20%-25% A at 0-20 min, 25%-33% A at 20-30 min, 33%-50% A at 30-55 min, 50%-60% A at 55-65 min, and 60% A between 65 min and 80 min, and peaks were detected at 280 nm. The fourteen compounds were assigned by HPLC-Orbitrap MS methods. The regression coefficient for the linear equations for the 14 compounds ranged between 0.9998 and 1. The limits of detection and quantification lay in the range of 0.032-2.461 and 0.154-8.202 μg·mL(1), respectively. The relative recovery rates for the 14 compounds were in the range of 93.90%-106.73% with RSDs being less than 5%. Coefficient variations for intra-day and inter-day precisions were in the range of 0.27%-2.38% and 0.31%-3.51%, respectively. In summary, the validated method was applied to the simultaneous determination of the 14 components in 29 different licorice samples and was proven to be suitable for quality evaluation of licorices and their active fractions.
Subject(s)
Chromatography, High Pressure Liquid , Methods , Flavonoids , Glycyrrhiza , Chemistry , Glycyrrhizic Acid , Mass Spectrometry , Methods , Reproducibility of ResultsABSTRACT
The marine biological source of mineral drugs recorded in Chinese Pharmacopoeia (2010 version) mainly including pearl, nacre, clam shell, common oyster shell, ark shell, cuttle bone, and sea-ear shell are widely used in clinical. Calcium carbonate and a small amount of protein are the main components in this type of drugs. In this paper, a systematical and comparable study were carried out by determination of calcium carbonate by EDTA titration method, the crystal of calcium carbonate by X-Ray powder diffraction and the total amino acids (TAAs) of the hydrolyzed samples by ultraviolet spectrophotometry method. As a result, the crystal structure is calcite for common oyster shell, mixture of calcite and aragonite for nacre and sea-ear shell, aragonite for the other drugs. The content of calcium carbonate ranged from 86% to 96%. Cuttle bone has the highest amount of TAAs among the seven drugs which reached 1.7% while clam shell has the lowest content of 0.16% on average. In conclusion, an effective method was developed for the quality control of marine mineral drugs by comprehensive analysis of calcium carbonate and TAAs in the seven marine mineral drugs.
Subject(s)
Animals , Amino Acids , Chemistry , Animal Shells , Chemistry , Calcium Carbonate , Chemistry , Crystallization , Edetic Acid , Chemistry , Mollusca , Chemistry , Classification , Pharmaceutical Preparations , Chemistry , Reference Standards , Quality Control , Reproducibility of Results , Seawater , Species Specificity , Spectrophotometry, Ultraviolet , X-Ray DiffractionABSTRACT
<p><b>OBJECTIVE</b>To study the biotransformation of podophyllotoxin by the cell suspension culture and root culture systems of Rheum palmatum.</p><p><b>METHOD</b>Using plant tissue culture technology and HPLC techniques to isolate products. The structures were elucidated by spectroscopic means.</p><p><b>RESULT</b>Cell suspension culture of R. palmatum could convert podophyllotoxin to produce picropodophyllotoxin with the yield of 73.8%, while root culture of R. palmatum could convert podophyllotoxin to produce epipodophyllotoxin and apopodophyllotoxin.</p><p><b>CONCLUSION</b>Podophyllotoxin did not affect the pH value of the media used in tissue cultures. Both cell suspension culture and root culture of R. palmatum can convert podophyllotoxin.</p>
Subject(s)
Chromatography, High Pressure Liquid , Molecular Structure , Plant Roots , Metabolism , Podophyllotoxin , Chemistry , Metabolism , Rheum , Metabolism , Tissue Culture Techniques , MethodsABSTRACT
<p><b>OBJECTIVE</b>To establish fingerprinting of Flos Buddleja by using RP-HPLC for the quality control.</p><p><b>METHOD</b>The HPLC condition was as follows: Inertsil ODS-3 C18 analytical column (4.6 mm x 250 mm, 5 microm), gredient eluation with MeCN (0.1% TFA)-H2O (0.1%TFA), flow rate 1.0 mL x min(-1), detection wavelength 254 nm. 10 commercial samples were analyzed to establish a fingerprinting.</p><p><b>RESULT</b>Among the obtained fingerprinting, most of the detected peaks were separated effectively. The accuracy, repeatability and stability of this method were satisfied. The RSDs of relative retention time and area of aimed peaks which existed in all samples wereless than 5%. Theresults were in accordance with the request of fingerprinting.</p><p><b>CONCLUSION</b>The established fingerprinting can be used for the quality control of Flos Buddleja.</p>