Post-Chromatographic Derivatization Coupled with Mass Spectrometry as a Method of Profiling and Identification of Antioxidants; Ligustrum vulgare Phytocomplex as an Example.

High-performance liquid chromatography (HPLC) and high-performance thin-layer chromatography (HPTLC) coupled with radical scavenging assays, such as 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) can be both used for the detection of the antioxidants in plant extracts. In this study, the ethanolic (70% v/v) extracts from different morphological parts of Ligustrum vulgare collected at different stages of maturity were used as the source of antioxidants. The final identification of antioxidants was performed using high-resolution mass spectroscopy (HRMS). As a result, 19 compounds with antioxidant properties detected with HPLC-ABTS assay and 10 compounds detected with HPTLC-DPPH/ABTS assay were identified, mostly from the group of iridoids, phenylethanoids, and flavonoids. When comparing different L. vulgare samples, it was found that the extracts obtained from leaves contained the greatest number of antioxidants. The results of this study suggest that HPTLC-DPPH/ABTS as well as HPLC-ABTS derivatization coupled with the HRMS can be successfully used for profiling and identification of antioxidants from natural sources. Planar chromatography is more suitable for screening multiple samples because of its simplicity, whereas more challenging liquid chromatography provides more detailed information and is therefore better for a selected set of samples.

Phytochemical screening and biological evaluation of Greek sage (Salvia fruticosa Mill.) extracts.

This study explores the influence of extraction solvents on the composition and bioactivity of Salvia fruticosa extracts. Ultrasound-assisted extraction with water, ethanol and their mixtures in variable proportions was used to produce four different extracts. An untargeted UPLC/MS­based metabolomics was performed to discover metabolites profile variation between the extracts. In the analyzed samples, 2704 features had been detected, of which 95 were tentatively identified. The concentrations of the important metabolites, namely, caffeic acid, carnosic acid, carnosol, rosmarinic acid, salvianolic acid B and scutellarin, were determined, using UPLC-PDA methods. Rosmarinic acid was the dominant metabolite and antioxidant in all tested extracts, except the aqueous extract, in which scutellarin was the most abundant compound. The extracts and standards were examined for antioxidant activity and xanthine oxidase (XO) inhibitory activity. The most diverse in terms of chemical composition and rich in antioxidant compounds was 70% ethanolic extract and the strongest antioxidant was caffeic acid. All analyzed extracts showed the ability to inhibit XO activity, but the highest value was recorded for 30% ethanolic extract. Among tested standards, the most potent XO inhibitor was caffeic acid. The results suggest that the leaves of Greek sage are a source of natural XO inhibitors and may be an alternative to drugs produced by chemical synthesis.

Comparative Study on Assisted Solvent Extraction Techniques for the Extraction of Biologically Active Compounds from Sideritis raeseri and Sideritis scardica.

The plants in the Sideritis genus are postulated to exhibit several important medicinal properties due to their unique chemical composition. To isolate the targeted phytochemical compounds, the selection of a suitable extraction method is of primary importance. In this work, a comparative study on the phytochemical profiles of various Sideritis raeseri and Sideritis scardica extracts has been carried out. An untargeted metabolomics approach based on ultra-high performance liquid chromatography coupled with high-resolution mass spectrometry was applied to investigate the metabolic differences between extracts obtained by conventional extraction and extractions assisted by microwaves, ultrasounds and high pressure. Additionally, the influence of extraction solvents on HPLC antioxidant profiles obtained following the derivatization of analytes with ABTS reagent was evaluated. A total of 102 metabolites have been putatively identified. The major secondary metabolites groups were classified as flavonoids, terpenoids, phenylethanoid glycosides and phenolic acids. The main antioxidants in the extracts were isoscutellarein and hypolaetin derivatives as well as verbascoside and chlorogenic acid. The results showed that 70% ethanol was the most effective extractant for different classes of phytochemicals including antioxidants. In addition, extraction supported with microwaves, ultrasounds or high pressure improved the overall recovery of metabolites by about 3 times compared to the conventional extraction method.

Comparative evaluation of different methods for determining phytochemicals and antioxidant activity in products containing betalains - Verification of beetroot samples.

This study presents methods that can be used to assess the health quality of products containing betalains. The paper compares and verifies data on the phytochemical composition of three different pigmented beetroot cultivars using spectrophotometric, HPLC-DAD, HPTLC and LC-Q-Orbitrap-HRMS techniques. Additionally, we compared the total antioxidant activity in both the cell-free and cellular systems. Betalain contribution to antioxidant activity was also determined using post-column derivatization and it was found that in the case of red beetroot it is about 50%. Photometric measurements are recommended for a simple and inexpensive analysis of the total betacyanin and betaxanthin content. Liquid chromatography techniques produced more precise information on phytochemical composition in the tested samples. The combination of liquid chromatography with high-resolution mass spectrometry produced the largest amount of quantitative and qualitative data; in beetroot samples sixty-four phytochemicals have been identified therefore, this approach is recommended for more detailed metabolomics studies.