Untargeted Metabolomics Based Prediction of Therapeutic Potential for Apigenin and Chrysin

Abstract

The prominent flavonoids apigenin and chrysin have been demonstrated to have systemic benefits. Our previous work was first to establish the impact of apigenin and chrysin on cellular transcriptome. In the current study, we have revealed the ability of apigenin and chrysin to alter the cellular metabolome based on our untargeted metabolomics. Based on our metabolomics data, both these structurally related flavonoids demonstrate diverging and converging properties. Apigenin demonstrated the potential to possess anti-inflammatory and vasorelaxant properties through the upregulation of intermediate metabolites of alpha-linolenic acid and linoleic acid pathways. Chrysin, on the other hand, exhibited abilities to inhibit protein and pyrimidine synthesis along with downregulation of gluconeogenesis pathways based on the altered metabolites detected. Chrysin-mediated metabolite changes are mostly due to its ability to modulate L-alanine metabolism and the urea cycle. On the other hand, both the flavonoids also demonstrated converging properties. Apigenin and chrysin were able to downregulate metabolites involved in cholesterol biosynthesis and uric acid synthesis, namely 7-dehydrocholesterol and xanthosine, respectively. This work will provide understanding regarding the diverse therapeutic potential of these naturally occurring flavonoids and help us in curbing an array of metabolic complications.

Keywords: apigenin; chrysin; flavonoids; metabolomics; therapeutic prediction.

Figure 1

IUPAC nomenclature for structurally related flavonoids. (A) Apigenin is a 5,7-Dihydroxy-2-(4′-hydroxyphenyl)-4H-chromen-4-one and (B) chrysin is a 5,7-Dihydroxy-2-phenyl-4H-chromen-4-one.

Figure 2

Pathway enrichment analysis of altered metabolites in mouse embryonic fibroblasts following apigenin treatment. Following 24 h of apigenin treatment, there were numerous metabolite pathways, which were altered in negative ion mode.

Figure 3

The figure depicts pathway enrichment analysis and the metabolic pathways that were significantly altered in positive ion mode following apigenin treatment.

Figure 4

Analysis of components of the alpha-linolenic acid pathway regulated by apigenin. The metabolites belonging to the alpha-linolenic acid pathway that appeared in the analysis panel were: (A) eicosapentaenoic acid, (B) docosapentaenoic acid, and (C) docosahexaenoic acid. The raw values plotted on the upper inserts and the raw values converted to a log2 fold change and plotted using GraphPad Prism were used in the main figure. Significance was determined based on * p < 0.05 and with a log2 fold change difference of +/− 1.5 fold change.

Figure 5

Metabolites of the linoleic acid pathway regulated by apigenin. In the linoleic acid pathway, the metabolites that were differentially regulated by apigenin were: (A) arachidonic acid, and (B) adrenic acid. The upper inserts represent the raw values. The log2 fold change values were plotted using GraphPad and significance set at * p < 0.05 with a +/− 1.5 fold change.

Figure 6

Metabolic pathways regulated by chrysin treatment in negative ion mode. The pathway enrichment analysis and enrichment ratio in the negative ion mode are illustrated in this figure.

Figure 7

Chrysin-regulated pathways in positive ion mode. The enrichment pathway analysis along with the enrichment ratio in the positive ion mode are clearly depicted.

Figure 8

Regulation of alanine metabolism by chrysin: The schematics for alanine regulation in non-hepatic cells are represented. (A) The source of alanine is from either of the glucose metabolites—pyruvate or lactate. The formed alanine is then effluxed of the cell. (B) Represents L-alanine raw values (upper insert) and log2 fold change values. The log2 fold change values for pyruvic acid and lactic acid, which are the acidic forms for pyruvate and lactate with their respective raw values (inserts), are represented in Figure 3 panel (C,D), respectively * p < 0.05.

Figure 9

Chrysin-mediated regulation of metabolites in the urea cycle: Based on the metabolite panel, the urea cycle was the other top regulated metabolic pathway. (A) Glutamate and acetyl CoA act as major substrates for the formation of N-acetyl glutamate and finally carbamoyl phosphate, which is the precursor for substrates in the urea cycle including citrulline, arginine, and ornithine. The raw values (upper inserts) and the log2 fold change values of (B) L-arginine, (C) D-ornithine, and (D) orotidine are expressed, respectively. The protonated forms of N-acetylated glutamate and glutamate, N-acetyl L-glutamic acid, and L-glutamic acid with the raw values (upper inserts) and calculated log2 fold change values are represented in (E,F), respectively * p < 0.05.

Figure 10

Downregulation of cholesterol and uric acid pathways by apigenin and chrysin: Both apigenin and chrysin demonstrated to have a suppressive effect on the cholesterol biosynthetic pathway and uric acid pathway based on the major metabolites belonging to these pathways. (A) 7-Dehydrocholesterol and (B) xanthosine levels as determined by raw values (upper inserts) and log2 fold change values as expressed in respective figures with a significance of * p < 0.05.

Figure 11

Summary diagram demonstrating the distinct and similar metabolic pathways regulated by apigenin and chrysin. Apigenin, through the regulation of the alpha-linolenic and linoleic acid pathways, generates endogenous anti-inflammatory and vasorelaxant metabolites. On the other hand, chrysin, by regulating alanine and urea cycles, suppresses the gluconeogenesis, protein synthesis, and pyrimidine synthesis pathways. Both apigenin and chrysin demonstrate converging effects by inhibiting the generation of metabolites involved in biosynthesis of cholesterol and uric acid pathways.