MOL2NET, International Conference Series on Multidisciplinary Sciences UHPLC-HRMS analysis of Coccoloba cowellii , an endemic endangered plant from Cuba

Abstract. Coccoloba cowellii Britton (Polygonaceae) is an endemic and critically endangered plant that only grows in Camagüey, province of Cuba. In this study a total of 13 compounds were identified in a methanolic

https://mol2net-07.sciforum.net/Introduction Cuba is recognized as the island with the highest degree of endemism in the West-Indies, including more than half of its plant species [1].Since plants in Cuba are frequently subjected to harsh environmental conditions (e.g., high temperature, drought, high levels of sunlight, salinity, nutrient-poor soil conditions…), combined with the fact that Cuban flora is pharmacologically and chemically underinvestigated [2], the development of conservation strategies to preserve the plant species of the island is necessary.The genus Coccoloba comprises approximately 120-150 species of flowering plants from the subfamily Erigonoideae of the Polygonaceae family, order Caryophyllales.It is native to the tropical and subtropical regions of America, i.e.South America, the Caribbean and Central America, with two species that extend to Florida [3].A small number of species of Coccoloba are used in traditional medicine in tropical and subtropical regions of the Americas related to the treatment of several ailments [4][5][6].The phytochemistry of the genus has not widely been explored, and most of the studies are centered on the more common species C. uvifera (sea grape, native to coastal beaches throughout tropical America and the Caribbean).In Cuba, the presence of 34 species of Coccoloba has been reported [7].From them, 25 are recognized as endemic.One of the almost unknown endemic species of this genus that grows in Cuba is Coccoloba cowellii Britton, which classifies as critically endangered (CR) according to the International Union for Conservation of Nature (IUCN) [8].Ultrahigh-performance liquid chromatographyhigh resolution mass spectrometry (UHPLC-HRMS) was selected as analytical technique suitable for studying the nonvolatile phytochemical composition of C. cowellii leaves, collecting as little plant material as possible.

Plant material collection and processing
Leaves of Coccoloba cowellii were collected near to Albaisa, in the municipality of Camagüey (Lat.21.43615, Long.-77.83253),Cuba.The plant material was taxonomically identified by the curator of "Julián Acuña Galé" herbarium at the University of Camagüey (HIPC, http://sweetgum.nybg.org/science/ih/herbarium-details/?irn=124935), where a voucher specimen was deposited (number 12057).The plant material (0.35 kg of fresh leaves), after cleaning, was dried at room temperature until constant weight and subsequently ground using a mill.The dried leaves (0.25 kg) were defatted with n-hexane and later on exhaustively stirring macerated in 250 mL of 80% methanol/water mixture (v/v) at room temperature during five days.Every 24 h, the solvent was collected and the material macerated with other 250 mL.The filtrate was concentrated using a rotary evaporator under reduced pressure below 40 °C.The resulting reduced filtrate was freeze dried, yielding 25.07 g dry total extract and stored at -20 °C until further use.

UHPLC-HRMS analysis
The analysis of the Coccoloba cowellii extract was carried out using a LC-HRMS method according to Bijttebier et al., 2016 [9] and Baldé et al., 2020 [10].For the HPLC-DAD-QTOF analyses, accurate mass measurements were done using a Xevo G2-XS QTOF spectrometer (Waters, Milford, MA, USA) coupled with an ACQUITY LC system equipped with MassLynx version 4.1 software.Data were recorded using MS E in the positive and negative ionization modes (two analyses per mode), and a ramp https://mol2net-07.sciforum.net/collision energy from 10 to 30 V was applied to obtain additional structural information.Leucineencephalin was used as the lock mass.UV detection was performed at 360 nm.

Data processing
The HPLC-MS raw data were converted to abf files (ReifycsAbf Converter) and processed with MS-DIAL version 4.24 [11].The alignment results were exported using the GNPS export function of MS-DIAL.A molecular network was created with the Feature-Based Molecular Networking (FBMN) workflow [13] on the Global Natural Products Social (GNPS) molecular networking web-platform (https://gnps.ucsd.edu)[14].The data were imported into Cytoscape v3.7.2 (The Cytoscape Consortium, New York, NY, USA) for visualization.

UHPLC-HRMS analysis
A qualitative analysis of the chemical composition of C. cowellii leaves was carried out using UHPLC-UV-QTOF-ESI-MS in negative ionization mode.Figure 1 shows the base peak intensity (BPI, peaks 1 to 15 corresponding to Table 2) chromatogram at 280 nm (a) and in MS negative ionization mode (b) of C. cowellii leaf extract.From the peak intensity of the UV chromatogram (Figure 1a), is inferred that compound 6 (Rt=10.60min) appears as main compound.Peaks 7, 12 and 13 (Rt=10.85,12.02 and 12.37) also reach high concentration ratios regarding the rest of compounds.https://mol2net-07.sciforum.net/A dereplication strategy was used to analyze the raw data obtained.With this purpose, the spectra in the network were searched and matched with GNPS spectral libraries rendering 12 library hits (Table 1).The molecular networking job can be publicly accessed at https://gnps.ucsd.edu/ProteoSAFe/status.jsp?task=a2f9e6e25ca64043a36a3d2fb09270c5.The matched compounds were mainly glycosides and glucuronides of the aglycones quercetin and myricetin, proanthocyanidins and one methoxylated flavonoid.MZErrorPPM: ppm error with the spectral library match, LibMZ: m/z value of the spectral library match.
Later on, all the major peaks detected were tentatively characterized by means of MS data, together with the interpretation of the observed MS/MS spectra in comparison with those found in the literature and the information derived from the FBMN analysis previously done and MS-DIAL software results.The formerly identified phytochemicals from the same botanical family or species were also utilized in the identification when applicable.This analysis allowed the identification of 13 phytochemical compounds from a total of 15 peaks.Four compounds were confirmed using authentic standards while the others were tentatively characterized.All of them were reported for the first time in C. cowellii leaves (Table 2).

Table 1 .
Library hits found in the spectra of the methanolic extract of C. cowellii against the GNPS database.

Table 2 .
Chemical composition of the total extract from the leaves of C. cowellii.