Isolation, separation, identification, and quantification of bioactive methylated flavone regioisomers by UHPLC-MS/MS.

Hafiza Noreen,James S O Mccullagh,Edward N Smith,Muhammad Farman,Tim D W Claridge

doi:10.1002/ansa.202100016

Abstract

Methylated flavones, commonly found in many plants of the Brassicaceae family, have potent antioxidant and anticancer activity with diverse therapeutic potential. However, the specific regioisomers of methylated flavones can have significantly different biochemical and potentially therapeutic properties as shown by various bioassays but analytically differentiating these compounds has been technically challenging and rarely reported. In this study, we demonstrate differentiation and identification of selected bioactive methylated flavone regioisomers, namely 5,7,3'-trihydroxy-4'-methoxyflavone, and 5,7,4'-trihydroxy-3'-methoxyflavone extracted from Coronopus didymus, a member of the Brassicaceae family, using ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (UPLC-ESI-QTOF-MS/MS). Characteristic MS/MS product ions produced from neutral loss of carbon monoxide, and a methyl radical from the [M-H]- ion, exhibited differential relative abundances attributed to different structural stabilities under the same activation and collision-induced dissociation conditions. MS/MS also provided structural information which was sufficient to differentiate the methylated regioisomers and determine the position of the methyl group based on interpretation of their respective fragmentation patterns. Quantification showed 5,7,4'-trihydroxy-3'-methoxyflavone was at least 1.60mg per 10g plant material in C. didymus extracts. This study demonstrates a straightforward and novel approach to rapidly differentiate, identify and quantify regio-isomeric methylated flavone natural products using reversed-phase UPLC-MS/MS.

Highlights

IntroductionFlavonoids constitute a very large and important group of polyphenolic natural products present in almost all plant species
ABBREVIATIONS: C. didymus, coronopus didymus; CID, collision-induced dissociation; Da, dalton; LLOD, lower limit of detection; LLOQ, lower limit of quantification; UHPLC-Mass spectrometry (MS)/MS, ultra-high performance liquid chromatography-tandem mass spectrometryFlavonoids constitute a very large and important group of polyphenolic natural products present in almost all plant species
We reported that the regioisomeric methylated flavone (5,7,4′-trihydroxy-3′-methoxyflavone) with 3′-OCH3, isolated from Coronopus didymus Linn., had 45 times more potent cytotoxic activity against HeLa cells and 9 times more cytotoxic against LN18 cancer cells, compared to its regioisomer (5,7,3′-trihydroxy-4′-methoxyflavone) with 4′-OCH3.16 C. didymus is enriched in a large number of volatile phytochemicals and methylated flavone regioisomers with high antioxidant and cytotoxic activity.[16,17]

Summary

Introduction

Flavonoids constitute a very large and important group of polyphenolic natural products present in almost all plant species. Diosmetin (5,7,3′-trihydroxy-4′-methoxyflavone) has been shown to possess chemo-preventive, anti-mutagenic, anti-allergic, anti-osteoporosis,[4] anti-pigmentation,[5] iron-chelating, and antioxidant activity.[6] Diosmetin has been used for the treatment of ceruleininduced acute pancreatitis in mice.[7] The diosmetin and luteolin have been shown to exert synergistic cytostatic effects in human hepatoma (HepG2) cells.[8]. The regioisomer, chrysoeriol (5,7,4′-trihydroxy-3′-methoxyflavone) possesses antioxidant and anti-inflammatory,[9] antitumor,[10] antimicrobial, and selective bronchodilator effect.[11] It potentially serves as a novel cardioprotective agent against doxorubicin-induced cardiotoxicity.[12] Chrysoeriol has shown antitumor activity against human multiple myeloma cells in vitro[13] and modulates the downstream signal transduction pathways of platelet-derived growth factor, for the treatment of vascular diseases and protects osteoblasts from oxidative stress-induced toxicity.[14,15]

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Conclusion