Optimization for quantification of sorghum tannins by Ferric ammonium citrate assay

Due to the complexity of structure,it is difficult to determine the content of sorghum tannins.The current method for the determination of sorghum tannins is the Ferric ammonium citrate assay described in ISO 9648.However,the standard tannic acid(TA)used in the ISO 9648 method is significantly different in both structure and dynamics from sorghum tannins,resulting in inaccurate quantitative result.Furthermore,the extraction solvent,75%dimethylformamide(DMF),used in each step of this method had large toxicity to human.Hence,the objective of this study is to optimize the ISO 9648 method by selecting proper standard and extraction solvents so as to enhance the accuracy and safety.In this study,Sephadex LH-20 chromatography combined with high pressure liquid chromatography was used to purify and identify the extracted sorghum tannin(ST).Then,commercial TA,epicatechin(EA),grape seed procyanidin(PA)were selected as the standard and ST was used as reference for Ferric ammonium citrate assay to compare the effects of the three standards.Furthermore,the extraction rates of sorghum tannins in the presence of several low toxic solvents,such as absolute ethanol,absolute methanol,70%ethanol solution,70%methanol solution and 70%acetone solution,were compared to determine the alternative solvents of 75%DMF solution.The results showed that PA was superior to TA and EA in structure,with satisfactory color yield close to ST,and higher accuracy than TA.In terms of the extraction solvent,70%acetone solution was selected to replace 75%DMF solution because of its higher extraction yield(only next to 75%DMF)and low toxicity.Verification experiment results showed that both the recovery rate and the repeatability of the optimized method met the requirements of AOCO.Moreover,the optimized method,with higher accuracy and safety than the ISO 9648 method,can be applied widely in laboratory.

Magnetic resonance imaging focused on the ferritin heavy chain 1 reporter gene detects neuronal differentiation in stem cells

The neuronal differentiation of mesenchymal stem cells offers a new strategy for the treatment of neurological disorders.Thus,there is a need to identify a noninvasive and sensitive in vivo imaging approach for real-time monitoring of transplanted stem cells.Our previous study confirmed that magnetic resonance imaging,with a focus on the ferritin heavy chain 1 reporter gene,could track the proliferation and differentiation of bone marrow mesenchymal stem cells that had been transduced with lentivirus carrying the ferritin heavy chain 1 reporter gene.However,we could not determine whether or when bone marrow mesenchymal stem cells had undergone neuronal differentiation based on changes in the magnetic resonance imaging signal.To solve this problem,we identified a neuron-specific enolase that can be differentially expressed before and after neuronal differentiation in stem cells.In this study,we successfully constructed a lentivirus carrying the neuron-specific enolase promoter and expressing the ferritin heavy chain 1 reporter gene;we used this lentivirus to transduce bone marrow mesenchymal stem cells.Cellular and animal studies showed that the neuron-specific enolase promoter effectively drove the expression of ferritin heavy chain 1 after neuronal differentiation of bone marrow mesenchymal stem cells;this led to intracellular accumulation of iron and corresponding changes in the magnetic resonance imaging signal.In summary,we established an innovative magnetic resonance imaging approach focused on the induction of reporter gene expression by a neuron-specific promoter.This imaging method can be used to noninvasively and sensitively detect neuronal differentiation in stem cells,which may be useful in stem cell-based therapies.

Dose-and time-dependentα-synuclein aggregation induced by ferric iron in SK-N-SH cells

Objective Intracellular formation of Lewy body （LB） is one of the hallmarks of Parkinson’s disease.The main component of LB is aggregatedα-synuclein,present in the substantia nigra where iron accumulation also occurs.The present study was aimed to study the relationship between iron andα-synuclein aggregation.Methods SK-N-SH cells were treated with different concentrations of ferric iron for 24 h or 48 h.MTT assay was conducted to determine the cell viability. Thioflavine S staining was used to detectα-synuclein aggregation.Results With the increase of iron concentration,the cell viability decreased significantly.At the concentrations of 5 mmol/L and 10 mmol/L,iron inducedα-synuclein aggregation more severely than at the concentration of 1 mmol/L.Besides,48-h treatment-induced aggregation was more severe than that induced by 24-h treatment,at the corresponding iron concentrations.Conclusion Ferric iron can induceα-synuclein aggregation,which is toxic to the cells,in a dose-and time-dependent way.