The qualitative composition and content of phenolic compounds in shoots of Casuarina equisetifolia L

The species Casuarina equisetifolia L. is widely used in forestry in many countries with a tropical climate. Extracts from the shoots of C. equisetifolia are known to be rich in phenolic compounds which play an important role in plant growth and development, as well as in adaptation to abiotic and biotic environmental factors. Additionally, they exhibit antiviral, antibacterial, anti-inflammatory, anti-tumor, neuroprotective, and other activities. In this study, the composition of phenolic compounds primarily consisting of monomeric ellagitannins was comprehensively investigated for the first time in the shoots of C. equisetifolia . The aim of this study was to investigate the composition and content of phenolic compounds in C. equisetifolia shoots using ultra performance liquid chromatography coupled with photodiode and mass spectrometric detectors (UHPLC-PDA-MS). The study focused on the green one-year-old photosynthetic shoots of the C. equisetifolia tree grown in the greenhouse of the All-Russian Institute of Medicinal and Aromatic Plants (VILAR, Moscow). Samples were collected in the first decade of July 2019. The shoots were frozen, lyophilized, and ground. A 15 mg specimens were extracted with 1 ml of 80 % acetone for 60 min at room temperature with constant stirring. The extract was centrifuged for 20 min at 14000 rpm and evaporated to dryness at 45 °C. The extraction was repeated two more times. The resulting dry extract was dissolved in 1 ml of deionized water for 60 min, centrifuged for 20 min at 14000 rpm, diluted five times with deionized water, and filtered. An ultra-high performance liquid chromatographic system (UHPLC, Acquity UPLC® 2.9.0, Waters Corporation, USA) with a photodiode array detector (190-500 nm) and triple quadrupole mass spectrometer (Xevo TQ, Waters Corporation, USA) was used for the analysis of phenolic compounds. Separation was carried out in an Acquity UPLC® BEH Phenyl column (2.1½100 mm, 1.7 µm, Waters Corporation, Ireland). Data analysis was performed using the DataAnalysis 4.0 software. Phenolic compounds were identified based on mass spectrometry data by determining the m/z value of the [M-H] ion and its m/z fragments. The content of different classes of phenolic compounds such as gallolyl-glucoses, ellagitannins, condensed tannins, flavonoids (quercetin and kaempferol derivatives) was determined using multiple reaction monitoring. The extract was found to contain 16 phenolic compounds, with 14 belonging to the class of hydrolyzable tannins and 2 to the class of flavan-3-ols. It was discovered that C. equisetifolia shoots accumulate monomeric ellagitannins with molecular masses ranging from 784 to 1068 Da, containing glucose as a polyol in either cyclic or linear form. Among the ellagitannins of C. equisetifolia , casuarinin, two isomers of pedunculagin, stachyurin, chebulic acid, casuarininin, and casuarictin were identified for the first time. Two compounds with a molecular mass of 1068 Da were preliminarily identified as isomers of pterocarinin A. Ellagic acid and its derivatives, ellagic arabinoside and ellagic rhamnoside, were also identified in shoots. The total content of phenolic compounds was 55 mg/g dry weight, with ellagitannins being the main phenolic compounds. Their content reached 42 mg/g, or 76 % of the total amount of all phenolic compounds. Galloyl-glucose and condensed tannins each accounted for 10 % of the total amount of all phenolic compounds. These findings suggest the potential use of C. equisetifolia shoots as a raw material for obtaining individual ellagitannins and studying their antiviral, anti-inflammatory, and anti-tumor activities.