Comparative metabolites profiling of different solvent extracts of Asparagus species cladodes using liquid chromatography-mass spectrometry-based metabolomics and molecular networking

Abstract

Introduction: Asparagus species are naturally distributed worldwide and are known for their pharmacological properties that offer cures for various ailments. However, the metabolic choreography of these Asparagus species is not well characterized, and the compounds contributing to their bioactivities remain unknown.

Objective: This study aimed to profile and compare the metabolomes of three Asparagus species cladodes using different solvent extractions.

Methods: An ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry-based metabolomics and molecular networking approach was used to study the effects of different solvents (ethyl acetate, methanol, and chloroform) with varying polarity on metabolites extraction and identification of bioactive compounds from three Asparagus species cladodes (Asparagus falcatus, Asparagus plumosus, and Asparagus densiflorus 'Meyersii').

Results: Multivariate statistical analyses (mainly principal component analysis) revealed a significant separation between the three solvents and the three species, indicating notable metabolic differences. A total of 118 metabolites were identified in the three species extracted with the different solvents, with methanolic and chloroform extracts containing more metabolites compared with ethyl acetate extracts. These metabolites were identified as belonging to the flavonoids, cinnamic acids, organooxygen compounds, steroids, fatty acids, benzenes, and glycerophospholipids compound classes. Furthermore, these compounds classes were differentially distributed among the three species, indicating chemical/chemotaxis differences between the compared species. Chloroform and methanol are recommended as the optimal solvents to obtain a high content of phytochemical compounds from Asparagus species cladodes.

Keywords: Asparagus; LC–MS; different solvents; metabolomics; molecular networking; phytochemical compounds.

FIGURE 1

(A–C) PCA score scatterplots indicating differences among different solvents (ethyl acetate [A], methanol [B], and chloroform [C]), where the color code represents samples within A. densiflorus ‘Meyersii’ (red), A. falcatus (green), and A. plumosus (blue). The model obtained was a two‐component model that explained 60.4% (A), 70.4% (B), and 75.4% (C) of the variation. (D–F) PCA score plot of A. densiflorus ‘Meyersii’ (D), A. falcatus (E), and A. plumosus (F). Datasets are initiated from ethyl acetate (green), methanol (blue), chloroform (red), and they indicate different clustering patterns. The model obtained was a two‐component model that explained 74% (D), 79% (E), and 79.6% (F) of the total variation.

FIGURE 2

An enhanced molecular network of Asparagus species cladodes ethyl acetate (A), chloroform (B), and methanolic extracts analyzed by liquid chromatography–tandem mass spectrometry (LC–MS/MS) using electrospray ionization in negative mode, with flavonoids, cinnamic acids, glycerophospholipids, and organooxygen compounds as the major metabolite class identities.

FIGURE 3

A zoomed‐in solvents molecular network of the glycerophospholipids class. Nodes from the selected molecular class are labeled with parent mass and displayed as pie charts that represent the distribution of the ion intensities of A. densiflorus ‘Meyersii’ (blue), A. falcatus (purple), and A. plumosus (yellow).

FIGURE 4

Zoomed‐in solvents molecular network of phenylpropanoids class (cinnamic acids [A] and flavonoids [B]). Nodes from the selected molecular class are labeled with parent mass and displayed as pie charts that represent the distribution of the ion intensities of A. densiflorus ‘Meyersii’ (blue), A. falcatus (purple), and A. plumosus (yellow).

FIGURE 5

A zoomed‐in solvents molecular network of steroids class. Nodes from the selected molecular class are labeled with parent mass and displayed as pie charts that represent the distribution of the ion intensities of A. densiflorus ‘Meyersii’ (blue), A. falcatus (purple), and A. plumosus (yellow).

FIGURE 6

Sunburst plot representing the classification of the annotated metabolites. The class of annotated metabolites include phenolics, steroids, organooxygen compounds, benzenes, and fatty acyls.

FIGURE 7

Metabolite distribution in Asparagus species. (A) A clustered averaged heatmap with a dendrogram showing hierarchical clustering on compound distributions. The color code on top indicates the species to which a column belongs: A. densiflorus ‘Meyersii’ (blue), A. falcatus (purple), and A. plumosus (yellow). (B–D) Distribution of compound classes in A. plumosus (B), A. falcatus (C), and A. densiflorus ‘Meyersii’ (D). Numbers within the pie charts indicate the total number of compounds of the class annotated in this species.

FIGURE 8

MetaboAnalyst pathway analysis. Metabolism pathways identified in Asparagus species (A). An indication of the flavone and flavonol biosynthesis pathway (B), glycerophospholipid pathway (C), and phenylpropanoid biosynthesis pathway (D). The abundance of each metabolite in three distinct species of Asparagus is depicted in pie charts.