Uncovering the therapeutic potential of green pea waste in breast cancer: a multi-target approach utilizing LC-MS/MS metabolomics, molecular networking, and network pharmacology

Abstract

BACKGROUND PISUM SATIVUM: (PS) is a universal legume plant utilized for both human and animal consumption, particularly its seeds, known as green peas. The processing of PS in food industries and households produces a significant amount of waste that needs to be valorized.

Methods: In this study, the metabolite profiles of the 70% ethanolic extracts of PS wastes, namely peels (PSP) and a combination of leaves and stems (PSLS), were investigated by liquid chromatography-electrospray ionization-quadrupole time-of-flight tandem mass spectrometry (LC-ESI-QTOF-MS/MS) followed by molecular networking.

Results: Different classes of metabolites were identified, being flavonoids and their derivatives, along with phenolic acids, the most abundant categories. Additionally, a comprehensive network pharmacology strategy was applied to elucidate potentially active metabolites, key targets, and the pathways involved in cytotoxic activity against breast cancer. This cytotoxic activity was investigated in MCF-7 and MCF-10a cell lines. Results revealed that PSLS extract exhibited a potent cytotoxic activity with a good selectivity index (IC_{50 =} 17.67 and selectivity index of 3.51), compared to the reference drug doxorubicin (IC_{50 =} 2.69 µg/mL and selectivity index of 5.28). Whereas PSP extract appeared to be less potent and selective (IC_{50 =} 32.92 µg/mL and selectivity index of 1.62). A similar performance was also observed for several polyphenolics isolated from the PSLS extract, including methyl cis p-coumarate, trans p-coumaric acid, and liquiritigenin/ 7-methyl liquiritigenin mixture. Methyl cis p-coumarate showed the most potent cytotoxic activity against MCF-7 cell line and the highest selectivity (IC_{50 =} 1.18 µg/mL (6.91 µM) and selectivity index of 27.42). The network pharmacology study revealed that the isolated compounds could interact with several breast cancer-associated protein targets including carbonic anhydrases 1, 2, 4, 9, and 12, as well as aldo-keto reductase family 1 member B1, adenosine A3 receptor, protein tyrosine phosphatase non-receptor type 1, and estrogen receptor 2.

Conclusion: The uncovered therapeutic potential of PSLS and its metabolite constituents pave the way for an efficient and mindful PS waste valorization, calling for further in-vitro and in-vivo research.

Keywords: Breast cancer; Green pea; LC-MS/MS metabolomics; Molecular networking; Network pharmacology; Waste valorization.

Fig. 1

Base peak chromatograms of Pisum sativum L. (green pea, PS) waste extracts in the negative ESI mode. (A) PSLS (leaves and stems extract) and (B) PSP (peels extract). Metabolite numbers are as listed in Table 1

Fig. 2

Base peak chromatograms of Pisum sativum L. (green pea, PS) waste extracts in the positive ESI mode. (A) PSLS (leaves and stems extract) and (B) PSP (peels extract). Metabolite numbers are as listed in Table 1

Fig. 3

Molecular network (showing clusters of metabolites of interest) based on LC-MS/MS data in Pisum sativum L. (green pea, PS) wastes. (i) Negative ESI mode and (ii) Positive ESI mode. Clusters A to F: flavonoids and their derivatives, G: fatty acids and their derivatives, H: saponins, I: amino acid and their derivatives. The network is displayed as a pie chart to reflect the relative abundance of each metabolite precursor ion in the analyzed samples

Fig. 4

Venn’s diagram representing the intersection of protein targets common to both the identified metabolites in Pisum sativum L. (green pea, PS) wastes and breast cancer

Fig. 5

(A) Compound-target-disease network and (B) sub-network showing the key metabolites and their interactions with the key protein targets

Fig. 6

PPI network of the most interconnected targets involved in breast cancer

Fig. 7

Functional analysis of the target genes identified by the compound-target-disease network. (A) Top GO biological processes, (B) top KEGG pathways, (C) top GO cellular components, and (D) top GO molecular functions. The circle size and the color density represent the number of involved genes and p-value, respectively. The fold enrichment is plotted on the abscissa while names are represented on the ordinate