Better than bench top. High speed antioxidant screening via the cupric reducing antioxidant capacity reagent and reaction flow chromatography

This study is based upon a recently established method for quantification of the antioxidant capacity of natural samples via a HPLC separation and a hyphenated selective detection (post-column derivatization with cupric reducing antioxidant capacity reagent) technique. This protocol demonstrated the main improvements to transform the quantitative protocol into a high-speed qualitative automated assay to screen samples for their potential total antioxidant capacity, typically performed via manual mixing of the sample and derivatisation and measured on a 96 well plate reader/bench top UV–Vis spectrophotometer. This approach with automated mixing is a more informative alternative for total antioxidant capacity as the antioxidant peaks are profiled for each sample within four minutes. This antioxidant profile may be used for routine analysis of raw materials and/or a guide for targeted approaches for structure elucidation for laboratories interested in early drug discovery, natural product research and the search of alternative antioxidant additives in consumer goods/therapeutics. This technique could also be used to monitor the stability, alteration or adulteration of manufactured goods containing antioxidants.Fil: Suktham, Thirada. University of Western Sydney; AustraliaFil: Jones, Andrew. University of Western Sydney; AustraliaFil: Acquaviva, Agustín. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Departamento de Química. Grupo Cromatografía; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; ArgentinaFil: Dennis, Gary R.. University of Western Sydney; AustraliaFil: Shalliker, R. Andrew. University of Western Sydney; AustraliaFil: Soliven, Arianne. University of Western Sydney; Australia. Universidad de la República; Urugua

Better than bench top : High speed antioxidant screening via the cupric reducing antioxidant capacity reagent and reaction flow chromatography

This study is based upon a recently established method for quantification of the antioxidant capacity of natural samples via a HPLC separation and a hyphenated selective detection (post-column derivatization with cupric reducing antioxidant capacity reagent) technique. This protocol demonstrated the main improvements to transform the quantitative protocol into a high-speed qualitative automated assay to screen samples for their potential total antioxidant capacity, typically performed via manual mixing of the sample and derivatisation and measured on a 96 well plate reader/bench top UV–Vis spectrophotometer. This approach with automated mixing is a more informative alternative for total antioxidant capacity as the antioxidant peaks are profiled for each sample within four minutes. This antioxidant profile may be used for routine analysis of raw materials and/or a guide for targeted approaches for structure elucidation for laboratories interested in early drug discovery, natural product research and the search of alternative antioxidant additives in consumer goods/therapeutics. This technique could also be used to monitor the stability, alteration or adulteration of manufactured goods containing antioxidants.Laboratorio de Investigación y Desarrollo de Métodos Analíticos (LIDMA

Total Antioxidant Capacity with Peak Specificity via Reaction Flow Chromatography and the Ferric Reducing Antioxidant Power Assay

An established ferric reducing antioxidant power (FRAP) assay was optimised by preparation of the derivatisation reagent in 300 mM formate instead of 300 mM acetate conditions, resulting in increased sensitivity signal to noise responses by up to five to ten times. The quantitative protocol for selective detection of antioxidants via a HPLC post column derivatisation (PCD) technique using the 300 mM formate FRAP reagent conditions was then transformed into a high-speed qualitative screening protocol by utilizing an emerging technology ‘reaction flow (RF) chromatography’. Reaction flow chromatography’s ability to screen for total antioxidant capacity with additional peak specificity/profile information of active peaks could be achieved in under 2 min.Laboratorio de Investigación y Desarrollo de Métodos Analíticos (LIDMA

Protein-based structures for food applications: from macro to nanoscale

Novel food structures' development through handling of macroscopic and microscopic properties of bio-based materials (e.g., size, shape, and texture) is receiving a lot of attention since it allows controlling or changing structures' functionality. Proteins are among the most abundant and employed biomaterials in food technology. They are excellent candidates for creating novel food structures due to their nutritional value, biodegradability, biocompatibility, generally recognized as safe (GRAS) status and molecular characteristics. Additionally, the exploitation of proteins' gelation and aggregation properties can be used to encapsulate bioactive compounds inside their network and produce consistent delivery systems at macro-, micro-, and nanoscale. Consequently, bioactive compounds which are exposed to harsh storage and processing conditions and digestion environment may be protected and their bioavailability could be enhanced. In this review, a range of functional and structural properties of proteins which can be explored to develop macro-, micro-, and nanostructures with numerous promising food applications was discussed. Also, this review points out the relevance of scale on these structures' properties, allowing appropriate tailoring of protein-based systems such as hydrogels and micro- or nanocapsules to be used as bioactive compounds delivery systems. Finally, the behavior of these systems in the gastrointestinal tract (GIT) and the impact on bioactive compound bioavailability are thoroughly discussed.JM and AP acknowledge the Portuguese Foundation for Science and Technology (FCT) for their fellowships (SFRH/BPD/89992/2012 and SFRH/BPD/101181/2014). This work was supported by Portuguese FCT under the scope of the Project PTDC/AGR-TEC/5215/2014, of the strategic funding of UID/BIO/04469 unit and COMPETE 2020 (POCI-01-0145-FEDER-006684), and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020—Programa Operacional Regional do Norte.info:eu-repo/semantics/publishedVersio

Green solvent selection and extractin protocol for selective recovery of anti-diabetic components from T. crispa

This study systematically explores a green extraction process of T. crispa stems to selectively isolate therapeutic compounds, borapetoside C (BPC) and magnoflorine (MGF), recognized for their anti-diabetic properties. In agreement with Hansen solubility parameters prediction, H2O and EtOH were found to be suitable solvents for extraction of BPC and MGF, respectively, resulting in 33 % and 45 % extractabilities after 60 min. Further investigations demonstrated considerable increase in BPC extractability using 20 % EtOH:H2O mixture at 40 °C, with complete extraction achieved after 60 min. While for MGF, pure-EtOH at 40 °C, was the most suitable solvent, showing the highest selectivity and complete extraction after 100 min. The BPC-rich extract from 20 % EtOH:H2O exhibited higher anti-diabatic activity (IC50 = 0.63 ± 0.03 and 0.72 ± 0.04 mg/mL, respectively, for α-glucosidase and α-amylase enzymes inhibition activity), and considerably lower cytotoxicity to L6 and HepG2 cells, with IC50 = 0.26 ± 0.16 and 0.24 ± 0.02 mg/mL, respectively, compared with the MGF-rich extract obtained with pure-EtOH. Based on these results, a sequential extraction scheme was proposed involving initial pure-EtOH extraction to selectively and completely remove MGF, followed by extraction with a 20 % EtOH:H2O mixture to recover the remaining BPC, which was approximately 80 % of the BPC originally present. The obtained MGF-free BPC-rich extract showed significantly lower cytotoxicity (IC50 = 0.31 ± 0.06 mg/mL against L6 cell) and higher enzyme inhibition activities (IC50 = 0.53 ± 0.32 and 0.52 ± 0.02 mg/mL for α-glucosidase and α-glucosidase enzymes inhibition activity), comparable to acarbose (IC50 = 0.43 ± 0.02 and 0.83 ± 0.03 mg/mL for α-glucosidase and α-glucosidase enzymes inhibition activity), the result that potentially leads to the development of a promising industrial process to harness T. crispa for diabetes prevention and treatment

Total antioxidant capacity with peak specificity via reaction flow chromatography and the ferric reducing antioxidant power assay

An established ferric reducing antioxidant power (FRAP) assay was optimised by preparation of the derivatisation reagent in 300 mM formate instead of 300 mM acetate conditions, resulting in increased sensitivity signal to noise responses by up to five to ten times. The quantitative protocol for selective detection of antioxidants via a HPLC post column derivatisation (PCD) technique using the 300 mM formate FRAP reagent conditions was then transformed into a high-speed qualitative screening protocol by utilizing an emerging technology ‘reaction flow (RF) chromatography’. Reaction flow chromatography’s ability to screen for total antioxidant capacity with additional peak specificity/profile information of active peaks could be achieved in under 2 min.Fil: Jones, Andrew. University of Western Sydney; AustraliaFil: Acquaviva, Agustín. Universidad Nacional de la Plata. Facultad de Cs.exactas. Laboratorio de Investigacion y Desarrollo de Metodos Analiticos. - Comision de Investigaciones Cientificas de la Provincia de Buenos Aires. Laboratorio de Investigacion y Desarrollo de Metodos Analiticos.; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; ArgentinaFil: Suktham, Thirada. University of Western Sydney; AustraliaFil: Dennis, Gary R.. University of Western Sydney; AustraliaFil: Shalliker, R. Andrew. University of Western Sydney; AustraliaFil: Soliven, Arianne. University of Western Sydney; Australia. Universidad de la República; Urugua

Information rich chromatographic separations of natural samples : the analysis of antioxidants in coffee using post column derivatisation and the CUPRAC assay on narrow bore reaction flow HPLC columns

In the present work, the CUPRAC PCD assay coupled to a narrow bore RF column (150 mm × 2.1 mm i.d., 5 µm particles) was tested on a variety of antioxidant standards and natural samples (coffee and decaffeinated coffee) to demonstrate the separation performance. As a result of being able to utilise narrow bore RF columns, several significant factors have been highlighted, including high resolution post column derivatisation assays and an approximate five-fold decrease in the volume of reagents (mobile phase and PCD reagents) resulting in a reduced cost of the analysis in terms of purchase price and waste disposal. Moreover, the CUPRAC antioxidant assay illustrates a variety of antioxidants in the coffee samples and also presents an extensive level of detection selectivity. Furthermore, the narrow bore RF PCD procedure reveals that caffeine was extracted from the coffee and yet a co-eluting antioxidant was not removed

Total antioxidant capacity with peak specificity via reaction flow chromatography and the ferric reducing antioxidant power assay

An established ferric reducing antioxidant power (FRAP) assay was optimised by preparation of the derivatisation reagent in 300mMformate instead of 300mMacetate conditions, resulting in increased sensitivity signal to noise responses by up to five to ten times. The quantitative protocol for selective detection of antioxidants via a HPLC post column derivatisation (PCD) technique using the 300 mM formate FRAP reagent conditions was then transformed into a high-speed qualitative screening protocol by utilizing an emerging technology ‘reaction flow (RF) chromatography’. Reaction flow chromatography’s ability to screen for total antioxidant capacity with additional peak specificity/profile information of active peaks could be achieved in under 2 min

A comparison of the performance of the cupric reducing antioxidant potential assay and the ferric reducing antioxidant power assay for the analysis of antioxidants using reaction flow chromatography

The cupric reducing antioxidant capacity (CUPRAC) assay is an established procedure used to measure antioxidant content. Recently, the CUPRAC assay was adapted for use in post column derivatisation coupled with high performance liquid chromatography (HPLC-PCD). In prior works reported in the literature, CUPRAC HPLC-PCD assays used large post column mixing coils leading to a deterioration in the observed separation efficiency. In this work we used the CUPRAC PCD assay with reaction flow (RF) chromatography. No reaction loop was required, thus preserving the efficiency of the separation. We subsequently compared the performance of the CUPRAC assay to that of an antioxidant assay that we have previously converted to RF chromatography, namely the ferric reducing antioxidant potential (FRAP) assay, both in RF mode. The relative response factor with respect to trolox was measured for both RF assays via the separation of a standard mixture of 16 compounds. Sample analysis of coffee and green tea via both RF assays was used to compare their ability to screen for bioactive candidates in complex natural products. In general, the CUPRAC assay is recommended as the RF PCD methodology of choice as it provided a higher level of performance compared to the FRAP assay, demonstrating less baseline noise interference, greater sensitivity, wider linear dynamic range and better assay precision