A 96-Well Plates-based UV Spectrophotometric Assay for Determination of Total Glucosinolates in Isatis indigotica Roots

Background: Glucosinolates (GLS) are important secondary metabolites in Cruciferae vegetables and herbs. Currently, the assays of total GLS determination are cumbersome (requiring acidic or enzymatic hydrolysis and addition of staining reagents), time-consuming, and indirect. High concentrations of inorganic salts are inevitably incorporated into the GLS products during separation. There is a need for a quantitative method for simple and rapid determination of total GLS after desalting process. Methods: A 96-well plates-based UV spectrophotometric method for determination of total GLS of Isatis indigotica roots was developed in the present study. The detection wavelength is set at 230 nm using quartz plates. This assay was validated using gluconapin and sinigrin as reference standards, and applied to determine the total GLS of I. indigotica roots prepared from five different desalting methods. Results: This assay is specific for total GLS prepared from I. indigotica roots, and it has acceptable accuracy (91.76–98.18% for quality control, and 95.59–102.52% for addition/recovery), precision (0.24–0.70% pooled RSD), reproducibility (0.31–1.84% RSD), and stability (0.24–1.45% RSD) over a 72-h period. Conclusion: The 96-well plates-based UV spectrophotometric assay is simple and accurate for high-throughput determination of total GLS.

Changes Occurring on the Activity of Salivary Alpha-Amylase Proteoforms in Two Naturalistic Situations Using a Spectrophotometric Assay

This study aimed to evaluate the changes in the activity of total salivary alpha-amylase (TsAA) and both the non-glycosylated and glycosylated salivary alpha-amylase proteoforms (NGsAA and GsAA, respectively) in physical and psychological stress models, estimated using a simple and easily set-up method. The method used was a spectrophotometric assay with 2-chloro-4-nitrophenyl-α-D-maltotriose (CNPG3) as a substrate, incubated with Concanavalin A (ConA) to remove most of the glycosylated protein from the sample. This method allowed the measurement of TsAA and estimation of NGsAA and GsAA activities with imprecision lower than 10%. When this method was applied to two different stress models, differences in the responses of the proteoforms were observed, with the NGsAA activity showing changes of higher magnitude after stress induction than the GsAA activity, and the highest correlation with the State–Trait Anxiety Inventory Scale in the Trier Social Stress Test (TSST). In conclusion, the activity of the two main sAA proteoforms can be easily estimated in saliva, and their measurement can provide additional information on TsAA activity in physical or psychological stress situations.

Experimental and Computational Evaluation of Chloranilic Acid as an Universal Chromogenic Reagent for the Development of a Novel 96-Microwell Spectrophotometric Assay for Tyrosine Kinase Inhibitors

The tyrosine kinase inhibitors (TKIs) are chemotherapeutic drugs used for the targeted therapy of various types of cancer. This work discusses the experimental and computational evaluation of chloranilic acid (CLA) as a universal chromogenic reagent for developing a novel 96-microwell spectrophotometric assay (MW-SPA) for TKIs. The reaction resulted in an instantaneous formation of intensely purple colored products with TKIs. Spectrophotometric results confirmed that the reactions proceeded via the formation of charge-transfer complexes (CTCs). The physical parameters were determined for the CTCs of all TKIs. Computational calculations and molecular modelling for the CTCs were conducted, and the site(s) of interaction on each TKI molecule were determined. Under the optimized conditions, Beer’s law correlating the absorbances of the CTCs with the concentrations of TKIs were obeyed in the range of 10–500 µg/well with good correlation coefficients (0.9993–0.9998). The proposed MW-SPA fully validated and successfully applied for the determination of all TKIs in their bulk forms and pharmaceutical formulations (tablets). The proposed MW-SPA is the first assay that can analyze all the TKIs on a single assay system without modifications in the detection wavelength. The advantages of the proposed MW-SPA are simple, economic and, more importantly, have high throughput.